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So heavier people generally have more blood than lighter people (this is why heavier people generally need to take higher doses of medication for the same effect of medication). They also have more blood to draw from. But this fails to differentiate between muscle mass and fat mass.
So here's my question: Is the net angiogenesis per gram of muscle more or less than the net angiogenesis per gram of fat?
Muscle is heavier than fat so actually it does. Muscle growth directly increases angiogenesis. Particularly the mechanism relies on hypoxia, any tissue with less than adequate blood supply secretes factors which result in angiogenesis. This is seen frequently when muscle is bulking as this increases the oxygen requirement but is also seen in heart muscle where subsequent to a heart attack tissue which isn't dead but mildly suffering caused new blood vessels to grow to supply this tissue (as the other ones are blocked which was the cause of the heart attack in most cases).
Autophagy, cancer and angiogenesis: where is the link?
Autophagy is a catabolic process for degradation of intracellular components. Damaged proteins and organelles are engulfed in double-membrane vesicles ultimately fused with lysosomes. These vesicles, known as phagophores, develop to form autophagosomes. Encapsulated components are degraded after autophagosomes and lysosomes are fused. Autophagy clears denatured proteins and damaged organelles to produce macromolecules further reused by cells. This process is vital to cell homeostasis under both physiologic and pathologic conditions.
While the role of autophagy in cancer is quite controversial, the majority of studies introduce it as an anti-tumorigenesis mechanism. There are evidences confirming this role of autophagy in cancer. Mutations and monoallelic deletions have been demonstrated in autophagy-related genes correlating with cancer promotion. Another pathway through which autophagy suppresses tumorigenesis is cell cycle. On the other hand, under hypoxia and starvation condition, tumors use angiogenesis to provide nutrients. Also, autophagy flux is highlighted in vessel cell biology and vasoactive substances secretion from endothelial cells. The matrix proteoglycans such as Decorin and Perlecan could also interfere with angiogenesis and autophagy signaling pathway in endothelial cells (ECs). It seems that the connection between autophagy and angiogenesis in the tumor microenvironment is very important in determining the fate of cancer cells.
Matrix glycoproteins can regulate autophagy and angiogenesis linkage in tumor microenvironment. Also, finding details of how autophagy and angiogenesis correlate in cancer will help adopt more effective therapeutic approaches.
A Vascular Endothelial Growth Factor-Dependent Sprouting Angiogenesis Assay Based on an In Vitro Human Blood Vessel Model for the Study of Anti-Angiogenic Drugs
Angiogenesis is the formation of new capillaries from pre-existing blood vessels and participates in proper vasculature development. In pathological conditions such as cancer, abnormal angiogenesis takes place. Angiogenesis is primarily carried out by endothelial cells, the innermost layer of blood vessels. The vascular endothelial growth factor-A (VEGF-A) and its receptor-2 (VEGFR-2) trigger most of the mechanisms activating and regulating angiogenesis, and have been the targets for the development of drugs. However, most experimental assays assessing angiogenesis rely on animal models. We report an in vitro model using a microvessel-on-a-chip. It mimics an effective endothelial sprouting angiogenesis event triggered from an initial microvessel using a single angiogenic factor, VEGF-A. The angiogenic sprouting in this model is depends on the Notch signaling, as observed in vivo. This model enables the study of anti-angiogenic drugs which target a specific factor/receptor pathway, as demonstrated by the use of the clinically approved sorafenib and sunitinib for targeting the VEGF-A/VEGFR-2 pathway. Furthermore, this model allows testing simultaneously angiogenesis and permeability. It demonstrates that sorafenib impairs the endothelial barrier function, while sunitinib does not. Such in vitro human model provides a significant complimentary approach to animal models for the development of effective therapies.
Keywords: Angiogenesis inhibitors DLL4 Human umbilical vein endothelial cell In vitro 3D model Microvessel Notch Sorafenib Sprouting angiogenesis Sunitinib Vascular endothelial growth factor.
Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
Concept of the present study:…
Concept of the present study: VEGF-induced angiogenesis-on-a-chip for gene and inhibitor study. (Top)…
Fabrication of the in vitro…
Fabrication of the in vitro human microvessel using a PDMS chip. (a) Schematic…
VEGF-induced sprouting from the established,…
VEGF-induced sprouting from the established, initial, microvessel. (a) Timeline of the method (arrows…
Knocking-down DLL4 affects sprouting angiogenesis…
Knocking-down DLL4 affects sprouting angiogenesis and microvessel integrity. (a) Phase-contrast images of microvessels…
Analysis of angiogenic sprouts using…
Analysis of angiogenic sprouts using optical coherence tomography. (a) 3D reconstructed images of…
Inhibition of sprouting angiogenesis by…
Inhibition of sprouting angiogenesis by sorafenib and sunitinib. (a) Timeline and schematic of…
The Role of miR-378a in Metabolism, Angiogenesis, and Muscle Biology
MicroRNA-378a (miR-378a, previously known as miR-378) is one of the small noncoding RNA molecules able to regulate gene expression at posttranscriptional level. Its two mature strands, miR-378a-3p and miR-378a-5p, originate from the first intron of the peroxisome proliferator-activated receptor gamma, coactivator 1 beta (ppargc1b) gene encoding PGC-1β. Embedding in the sequence of this transcriptional regulator of oxidative energy metabolism implies involvement of miR-378a in metabolic pathways, mitochondrial energy homeostasis, and related biological processes such as muscle development, differentiation, and regeneration. On the other hand, modulating the expression of proangiogenic factors such as vascular endothelial growth factor, angiopoietin-1, or interleukin-8, influencing inflammatory reaction, and affecting tumor suppressors, such as SuFu and Fus-1, miR-378a is considered as a part of an angiogenic network in tumors. In the latter, miR-378a can evoke broader actions by enhancing cell survival, reducing apoptosis, and promoting cell migration and invasion. This review describes the current knowledge on miR-378a linking oxidative/lipid metabolism, muscle biology, and blood vessel formation.
Cell metabolism governing the growth and functioning of each cell and a whole organism refers to chemical transformations and enzyme-catalyzed energy producing and energy utilizing reactions of carbohydrates, proteins, and lipids. Amongst the most metabolically active organs are liver, brain, gut, kidneys, and heart [1–3]. Although the rate of metabolic reactions is lower in skeletal muscles, they account for around 20% of the total energy expenditure due to a 50–60% contribution to a total body mass . Several microRNAs were reported to control processes related to metabolism such as insulin secretion (miR-9, miR-375), adipocyte differentiation (miR-143), fatty acid metabolism (miR-122), and myogenesis (miR-1, miR-133a, miR-133b, and miR-206) (reviewed in ). Of potential meaning is also miR-378a, located in the gene encoding master metabolic regulator, peroxisome proliferator-activated receptor gamma, coactivator 1 beta (PGC-1β) . miR-378a was found to affect lipid and xenobiotic metabolism, lipid storage, mitochondrial function, and shift towards a glycolytic pathway (Warburg effect) [5, 6]. Moreover, it affects muscle differentiation via regulation of myogenic repressor, MyoR . Because nutrients supply for metabolic processes is a matter of circulation, metabolically active tissues require high vascular density. Recently, miR-378a was reported to regulate tumor angiogenesis mainly via inhibition of tumor suppressors SuFu and Fus-1 [8, 9]. Thus, a growing body of evidence suggests a role of miR-378a as a mediator controlling reciprocally dependent processes such as metabolism, muscle differentiation/regeneration, and angiogenesis.
MicroRNAs (miRNAs miRs) are small noncoding RNA molecules with an average length of 21-22 nucleotides which can regulate gene expression posttranscriptionally by targeting mostly the 3′untranslated region (3′UTR) of mRNAs. However, miRNA target sites were also found on the 5′UTR regions of human mRNA . Since their discovery in C. elegans in 1993 , miRNAs currently can be recognized as potent players in wide spectrum of biological processes like development, differentiation, cellular defense mechanisms, and others. Conservative estimates state that over 30% of mRNA expression is regulated by miRNAs [12, 13]. However, others suggest that even up to 60% of the mRNA expression is targeted by miRNAs . miRNAs are often located in the introns of coding genes or noncoding sequences but can also be located in exons. Intronic miRNAs can be expressed together with their host gene mRNA being derived from a common RNA transcript [15, 16]. Other miRNAs can also have their own promoters, which enable independent expression, or can be organized in clusters sharing a common transcriptional regulation [17, 18].
miRNAs transcription is RNA polymerase II-dependent . In the case of miRNAs that are encoded in their own genes, the primary miRNA transcript (pri-miRNA) is several kilobases long, while miRNAs encoded in intronic regions of other genes (miRtrons) have shorter transcripts. The miRNA stem loop is excised from pri-miRNA by endoribonuclease drosha/DGCR8 (microprocessor complex) and a hairpin called pre-miRNA is exported from the nucleus by exportin-5 in a Ran-GTP dependent manner . An endoribonuclease dicer removes the hairpin loop sequence from pre-miRNA, creating a double stranded miRNA duplex. Depending on the relative stability of the miRNA duplex, one or, more rarely, both strands can be incorporated in a multiprotein RNA-induced silencing complex (RISC). When there is perfect pairing between the miRNA sequence and its target site, mRNA is cleaved by a protein part of the RISC called argonaute (AGO). If the pairing is partial, deadenylation of the mRNA via recruitment of the CCR4-NOT complex by the GW182 proteins inside the RISC takes place and the poly-A tail is lost, leaving the mRNA vulnerable to RNase activity, ubiquitination, and mRNA degradation. Alternatively, miRNA-induced RISC can also cause repression of translation by mechanisms such as, for example, the promotion of ribosome drop-off from the mRNA transcript or destabilization of the mRNA binding cap protein (Figure 1) (reviewed in [20, 21]).
3. miR-378a: Basics
miR-378a is embedded in the first intron of the ppargc1b gene encoding PGC-1β . The pre-miR gives rise to a leading strand (miR-378a-3p, previous IDs for murine sequence: mmu-miR-422b, mmu-miR-378, and mmu-miR-378-3p for human: hsa-miR-422b and hsa-miR-378) and a passenger strand (miR-378a-5p, previous IDs for murine sequence: mmu-miR-378, mmu-miR-37
, and mmu-miR-378-5p for human: hsa-miR-378 and hsa-miR-37 ). miRNA-378a-3p mature strand was first identified in 2004 in humans (originally named miR-422b) . Recently, other miRs with similar sequences but other localizations in the genome have been discovered and named: mmu-miR-378b,c,d in mouse and hsa-miR-378-b,c,d1,d2,e,f,g,h,i,j in human [23–27] (Table 1). In humans, miR-378a is by far the most expressed of the miR-378 sequences, with 7030 reads per million, in 78 experiments during deep sequencing, compared with 101–3220 reads per million, in 42–72 experiments for the other forms, respectively. In mice, miR-378a and miR-378b have similar expression levels, at 11700 and 11000 reads per million (miRBase, version 21, September 2015) . The sequence of miR-378a mature strands is highly conserved between species, with the miR-378a-5p strand being identical in both human and mice and the miR-378a-3p strand only differing in one nucleotide (Table 2) [6, 27].
PGC-1β may regulate several facets of energy metabolism such as mitochondrial biogenesis, thermogenesis, and glucose and fatty acid metabolism . Both strands of miR-378a are coexpressed with PGC-1β as shown, for example, in the liver and during adipocyte differentiation [6, 29]. The coexpression of miR-378a with its host gene implies they may share the same transcriptional activators, and miR-378a might be involved in similar processes as PGC-1β. Accordingly, high levels of (porcine) miR-378-1 (Table 2) expression are found in developing muscle, postnatal muscle, and myocardium and in brown adipose tissue [29, 30].
To date, only a limited number of miR-378a targets, which can be predicted based on in silico analysis, have been experimentally validated. The latter, however, imply a role of miR-378a in mitochondrial energy homeostasis, glycolysis, and skeletal muscle development and in tumor angiogenesis and other processes (Table 3).
|Critical regulator of the mitochondrial function||CYP2E1 |
|Involved in conversion of acetyl-CoA to glucose||cEBPα |
|Involved in the regulation of ATPase activity||ERRγ |
|Involved in control of oxidative metabolism||cEBPβ |
|Tyrosine kinase receptor, mediates the effects of IGF-1||GABPα |
|Orphan receptor, possibly involved in circadian rhythm||GDP |
|Involved in the lactic acid cycle||DDAH |
|Represses MyoD (and thus myogenesis)||VIM |
|Cytoskeletal protein anchoring position of organelles||Purβ |
|Nonmuscle α-actinin isoform, cytoskeletal protein||Actin |
|Regulatory protein highly expressed in muscle||Hsp70.3 |
|Suppressing cyclin D1||VEGF-A |
4. miR-378a in Metabolism
A major source of energy production comprises oxidation of glucose in glycolysis followed by oxidation of pyruvate in well-oxygenated cells (or followed by lactic acid fermentation in cancer, the Warburg effect) and from β-oxidation of lipids, which yields even more ATP per gram then carbohydrates metabolism. A complicated net of metabolic pathways requires advanced regulation by signaling molecules and hormones.
A location of miR-378a in the gene encoding PGC-1β  implies an involvement of miR-378a in metabolic pathways. Unlike its homologue, PGC-1α, the expression of PGC-1β is not elevated in response to cold exposure  but occurs in response to hypoxia, exercise, caloric restriction, or aging (reviewed in ). PGC-1β is preferentially expressed in tissues with relatively high mitochondrial content, such as heart, skeletal muscle, and brown adipose tissue . In 2002, PGC-1β was first cloned and shown to be upregulated in the liver during fasting . PGC-1β strongly activates hepatic nuclear factor 4 (HNF4) and PPARα, both of these nuclear receptors being important for the adaptation of hepatocytes to the effects of fasting. These findings could hint to a possible role of PGC-1β in the regulation of gluconeogenesis and fatty acid oxidation in the liver . PGC-1β is also involved in the regulation of energy expenditure or in the pathway of estrogen receptor-related receptors (ERRs) [33–37]. Since miRNAs originating in the introns of host genes may modulate the protein encoded by their parental genes and may be involved in the same mechanisms [38–40], miR-378a is proposed to be involved in the metabolic pathways affected by PGC-1β .
It was reported that mice lacking the first intron of the ppargc1b gene (and thus miR-378a) have a significantly higher oxygen capacity and mitochondrial function . Such mice also exhibit a resistance to high fat induced obesity. They identified a mediator complex subunit 13 (MED13), involved in nuclear receptor signaling, and carnitine acetyltransferase (CRAT), a mitochondrial enzyme involved in fatty acid metabolism, as targets of miR-378a-5p and miR-378a-3p, respectively . It implies that miR-378a plays a regulatory role in lipid metabolism. miR-378a-5p regulated also cytochrome P450 2E1 (CYP2E1) being involved in the metabolism of, for example, drugs and toxins .
In addition, it has been discovered that transcription factor nuclear respiratory factor-1 (NRF-1), a critical regulator of the expression of some important metabolic genes in mitochondria regulating cellular growth, is inhibited by miR-378a-3p . Thus, miR-378a can be considered as a regulator of mitochondrial function in cells overexpressing miR-378a.
Moreover, miR-378a-5p inhibits the mRNAs of ERRγ and GA-binding protein-α in breast cancer, which both interact with PGC-1β and together control oxidative metabolism . This leads to a reduction of tricarboxylic acid gene expression and oxygen consumption and an increase in lactate production, which shifts cells from an oxidative towards a glycolytic pathway. In this way, miR-378a-5p is believed to be a switch regulating the Warburg effect in breast cancer . Moreover, in situ hybridization experiments in this study showed that miR-378a-5p expression correlates with progression of breast cancer . The proposed regulating role of miR-378a-5p on the Warburg effect is in parallel with the effects of PGC-1β, which mediates gluconeogenesis and fatty acid metabolism after periods of fasting or intense exercise . Coactivation by PGC-1β of ERRα and PPARα makes muscle fibers in PGC-1β transgenic mice more rich in mitochondria and highly oxidative . Accordingly, such animals were able to run for longer times and at higher workloads .
Increased glycolytic rates and increased cell proliferation can be related to lactate production by lactate dehydrogenase (LDH). LDHA was found to be a direct target of miR-378a in the study of Mallat et al. . In this way, hsa-miR-378a-3p represses cell growth and increases cell death by targeting LDHA. Of note, hsa-miR-378a-3p and hsa-miR-378a-5p had opposite effects on LDHA expression. LDHA was significantly downregulated by miR-378a-3p overexpression and upregulated by miR-378a-5p overexpression .
In addition, miR-378a is also considered as an important factor in adipogenesis and lipid storage. There is a complex family of factors regulating those processes such as insulin , insulin-like growth factors (IGFs), glucagon, and thyroid hormones T3 and T4 (reviewed in [46–49]). As mentioned before, it was demonstrated that miR-378a-knockout mice do not get fat after 8 weeks of high fat diet . Such animals show an enhanced mitochondrial fatty acid metabolism and have elevated oxidative capacity of tissues targeted by insulin (e.g., liver, muscles, and adipose tissues) . In accordance with that, it was shown that mature strands of bta-miR-378-1 (Table 1) are expressed at higher level in cows with high (versus low) amount of back fat . Similarly, an inhibition of both mmu-miR-378a-3p and its host gene, PGC-1β, by the flavonoid fisetin lowered the accumulation of fat in the liver . Interestingly, mmu-miR-378a-5p was downregulated in mice that were fed a high fat diet for five months . In addition, miR-378a is highly induced during adipogenesis . Overexpression of miR-378a-3p/-5p during adipogenesis increased the transcriptional activity of CCAAT/enhancer-binding proteins (cEBP) alpha and beta, which can stimulate the expression of leptin, a hormone produced mainly by adipocytes which controls the homeostasis of body weight  (reviewed in [52, 53]). On the other hand, TNF-α, IL-6, and leptin are reported to increase the expression of miR-378a-3p in mature human adipocytes in vitro . These cytokines are mainly secreted in the adipose tissue and are suggested to be involved in development of insulin resistance [55, 56]. In addition, miR-378a-3p was shown to target insulin growth factor 1 receptor (IGF1R) and reduce the Akt signaling cascade in cardiomyocytes during cardiac development . Moreover, in tissues where IGF1 levels were high (e.g., fibroblasts and fetal hearts), miR-378-3p levels were very low, showing an inverse relation and suggesting a negative feedback loop between miR-378a-3p, IGF1R, and IGF1 .
As already mentioned, PGC-1β is a coactivator of PPARγ . The latter functions as a master regulator of adipogenesis and is involved in the formation of peroxisomes and the catabolism of very long chain fatty acids [58, 59]. PPARγ facilitates also the storage of fat in part by inhibiting leptin . Accordingly, the amount of adipose tissue does not increase in mice lacking PPARγ when they are fed a high fat diet . It was also reported that in cultured adipocytes mmu-miR-378a and PGC-1β expression is PPARγ, or rosiglitazone (a PPARγ ligand), dependent, finding two peroxisome proliferator response elements in the miR-378a loci . On the other hand, overexpression of miR-378a elevated the expression of PPARγ isoform 2 , suggesting positive feedback loop and confirming the involvement of miR-378a in the storage of fat.
There are several activators known to induce expression of PPARγ such as the members of the E2F transcription factor family and prostaglandin J-2 (PGJ-2) [63–65]. The latter may act through RAR-related orphan receptor alpha (RORA), which is frequently found in myocardium . In addition to PPARγ, RORA regulates also MyoD, a major transcription factor involved in skeletal muscle differentiation [67, 68]. Interestingly, RORA is a possible (but not yet validated) target for miR-378a-3p .
A proteomics-based study revealed several other proteins that are potentially targeted by rat miR-378a-3p or miR-378a-5p. miR-378a-3p was shown to regulate mannose-1-phosphate guanylyltransferase (GDP), dimethylarginine dimethylaminohydrolase 1 (DDAH1), and lactate dehydrogenase A (LDHA) all those proteins are participating in metabolic processes . On the other hand, tropomyosin beta chain, which is involved in the regulation of ATPase activity, was found to be a target of miR-378a-5p .
5. miR-378a in Muscle Development, Differentiation, and Regeneration
High levels of murine and rat miR-378a-3p, miR-378a-5p, and porcine miR-378-1 are reported in both developing and adult skeletal muscles [7, 30, 44]. miR-378a expression is enhanced during skeletal muscle differentiation .
MyoD and MyoG play a role in the processes of myogenesis and muscle regeneration, in which dormant satellite cells are activated upon muscle damage and start proliferating and differentiating into muscle fibers (reviewed in [70, 71]). It has been shown that miR-378a-3p targets the myogenic repressor MyoR during myoblast differentiation, which directly inhibits MyoD . On the other hand, MyoD is upregulated in response to miR-378a-3p overexpression and, conversely, the level of miR-378a-3p may be enhanced by MyoD . Thus, there is evidence for a feedback loop in which miR-378a-3p regulates muscle differentiation via inhibiting MyoR, leading to an increase of MyoD, which in turn enhances miR-378a-3p .
It has been suggested by Davidsen et al. that miR-378a may also control the development of skeletal muscle mass after training . In this study, miR-378a (strand not specified) was significantly downregulated in men who obtained low training-induced muscle mass gain compared to men who obtained high training-induced muscle mass gain .
A growing body of data shows a role of miR-378a-3p in the myocardium. miR-378a-3p is expressed mostly by cardiomyocytes, but not by nonmuscle cells, whereas the level of miR-378a-5p was reported to be very low in the heart . Fang et al. showed that miR-378-3p is significantly downregulated both in vitro in cardiomyocytes cell cultures exposed to hypoxia and in vivo during myocardial injury in rats . Overexpression of miR-378a-3p enhanced cell viability and inhibited apoptosis via caspase-3 inhibition . In contrast to this finding, another study found that miR-378a-3p downregulation enhanced the survival of cardiac stem cells via focal adhesion kinase activation and releasing connective tissue growth factor (CTGF), the latter being a target of miR-378a-3p . miR-378a inhibition enhanced cardiomyocytes survival after H2O2 treatment . Overexpression of miR-378a-3p in the study of Knezevic et al. increased apoptosis of cardiomyocytes via the direct targeting of IGF1R leading to a decrease of Akt signaling . This is in opposition to the previously mentioned study of Fang et al. which showed apoptosis was decreased during miR-378a-3p overexpression due to targeting of caspase-3 . The converse findings of the studies could be explained by different models used by Knezevic et al. and Fang et al. Because of those discrepancies, the role of miR-378a in apoptosis of cardiomyocytes requires further investigation. The finding that miR-378a-3p affects both IGF1R and the Akt pathway was confirmed  in a study which found that overexpression of miR-378a-3p in rhabdomyosarcoma suppressed IGF1R expression and affected phosphorylation of the Akt protein . miR-378a-5p was shown to target heat shock protein 70.3 (Hsp70.3) in mouse hearts in normoxic conditions, but in hypoxic conditions a transcript variant of Hsp70.3 without miR-378a-5p target site in its 3′-UTR is not repressed and can exert its cytoprotective properties .
Potential involvement of miR-378a in cardiac remodeling was also proposed. miR-378a-3p prevented cardiac hypertrophy by targeting either Ras signaling or the mitogen-activated protein kinase (MAPK) pathway [77, 78].
More studies on the effect of miR-378a expression in muscle disorders would also be desirable. In both Golden Retriever muscular dystrophy dogs and Duchenne muscular dystrophy patients, miR-378a expression was dysregulated, suggesting some relation between miR-378a expression and muscle dystrophy .
All in all, these findings suggest miR-378a-3p can be considered as an important player in cardiac development, remodeling, and hypertrophy.
6. miR-378a in Angiogenesis
Angiogenesis comprises development of new blood vessels from existing ones, regulated by cytokines and growth factors such as, for example, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and angiopoietin-1 (Ang-1). Their expression can be posttranscriptionally controlled by microRNAs such as miR-126, miR-296, miR-210, miR-21, and the miR-17
Skeletal muscles and heart muscle are tissues which, due to their oxygen and energy consumption, need to be sufficiently vascularized. One of the major regulators of angiogenesis is the hypoxia-inducible factor-1 (HIF-1), which controls over 100 genes  involved mainly in the glycolytic pathway and blood vessel formation, including VEGF-A or interleukin-8 [83–85]. VEGF is generally induced by hypoxia, while IL-8 in at least some cancers and endothelial cells can be diminished by HIF-1 via inhibition of c-Myc and Sp-1 transcription factors [86, 87]. c-Myc, known as a regulator of cell cycle progression, apoptosis, and cellular transformation, is also a potent activator of PGC-1β and, in turn, miR-378a-3p, upregulating their expression .
In addition, miR-378a has been shown to affect VEGF-A in two ways. Human hsa-miR-378a-5p (by the study of Hua et al. named as miR-378) can directly affect VEGF-A by competing with hsa-miR-125a for the same seed-region in the VEGF-A 3′UTR causing upregulation of VEGF-A . miR-378a-5p can also indirectly regulate VEGF-A affecting sonic hedgehog (SHH) signaling via Sufu inhibition, which is an inhibitory component of this signaling pathway . The SHH pathway in turn can upregulate VEGF-A and also other regulators of blood vessels formation, Ang-1 and Ang-2 expression [90–92]. Increased expression of VEGF-A, as well as PDGFβ and TGFβ1, was also seen in mesenchymal stromal cells (MSCs) transfected with rno-miR-378a-5p .
In skeletal muscles, VEGF-induced angiogenesis appears not to be regulated by the well-known HIF pathway but by PGC-1α, which coactivates estrogen-related receptor alpha (ERR-α) on binding sites in the promoter and the first intron of the VEGF gene, inducing its expression . This angiogenic pathway shows new roles for PGC-1α and ERR-α, which are important regulators of mitochondrial activity in response to stimuli like exercise. If there might be a role for PGC-1β in this pathway, it is yet to be examined. It is noteworthy, however, that miR-378a-5p is known to affect the estrogen receptors by inhibiting ERRγ, another estrogen-related receptor .
A role for miR-378a in cell cycle regulation and stimulation of cell growth is also proposed. In human mammary epithelial and breast cancer cell lines, miR-378a-3p can target the antiproliferative protein TOB2, which is a suppressor of cyclin D1, which in turn is required for cell cycle G1-phase to S-phase progression . Enhancing endothelial cell proliferation via cell cycle regulation contributes to the angiogenic process. Whether miR-378 affects endothelial cell proliferation by regulation of cell cycle remains to be established.
The role of miR-378a in the formation of blood vessels nourishing tumor and enabling tumor growth was revealed. miR-378a was found to be differentially regulated in different types of cancers  being downregulated in gastric cancer [96, 97], oral , and colon carcinoma , while being upregulated in renal  and lung cancer [9, 101]. Since it is also changed in serum or plasma of patients with prostate cancer , renal cancer [100, 103], and gastric cancer  and frequently found to be overexpressed in cryopreserved bone marrow mononuclear cells from acute myeloid leukemia patients , miR-378a might be considered as a biomarker.
The role of miR-378a in tumorigenesis, tumor growth, and tumor vascularization was revealed for the first time by Lee and coworkers in glioblastoma . They showed that miR-378a-5p enhances cell survival, reduces caspase-3 activity, and promotes tumor growth and angiogenesis, through repression of two tumor suppressors, Sufu and Fus-1 . Strikingly, nude mice injected with miR-378a-5p transfected cancer cells formed tumors of bigger volume and with larger blood vessels compared to GFP-transfected cells. On the other hand, high expression of miR-378a-5p in NSCLC correlated with brain metastases due to higher cell migration, invasion, and tumor angiogenesis . Another study confirmed the downregulation of Fus-1 by miR-378a-5p and showed that in the HepG2 liver cancer cells miR-378a-5p overexpression enhanced proliferation, migration, and, when injected in mice, invasion . Also in rhabdomyosarcoma, enhanced expression of miR-378a, VEGF, and MMP9 correlated with increased vascularization and metastasis . Taken together, these studies suggest that miR-378a may serve as a prognostic marker in cancer due to its effects on angiogenesis.
Our recent data confirmed the proangiogenic effect of miR-378a (both strands) in non-small cell lung carcinoma (NSCLC) and pointed at its correlation with heme-degrading enzyme, heme oxygenase-1 (HO-1). An involvement of HO-1 in angiogenesis and VEGF-A as well as IL-8 signaling was shown by us previously  however, its action in tumors seems to be complex . In NCI-H292 cell line overexpressing HO-1, miR-378a (both strands) levels decreased . Conversely, when HO-1 was silenced using siRNA, miR-378a expression was enhanced. Also overexpression of the miR-378a precursor sequence diminished HO-1 expression. Conditioned medium from NCI-H292 cells overexpressing miR-378a enhanced angiogenic potential of HMEC-1 endothelial cell line. Tumors formed by such cells in subcutaneous xenografts showed enhanced growth, vascularization, oxygenation, and distal metastasis in vivo . These interactions between miR-378a and HO-1 were confirmed in our studies on the role of the Nrf-2 transcription factor/HO-1 axis in NSCLC cell lines [110, 111].
On the other hand, enhanced expression of mmu-miR-378a-5p in 4T1 murine breast cancer cells decreased the proliferation, migration, and invasiveness of these cancer cells in vitro and in vivo by targeting fibronectin, resulting in inhibition of tumor growth .
Recent study showed that miR-378a may act as a biomarker for response to antiangiogenic treatment in ovarian cancer . Low expression of miR-378a was associated with longer progressive-free survival in patients with recurrent ovarian cancer treated with the antiangiogenic drug bevacizumab . Overexpression of the miR-378a precursor in ovarian cancer cells altered expression of genes associated with angiogenesis (ALCAM, EHD1, ELK3, and TLN1), apoptosis (RPN2, HIPK3), and cell cycle regulation (SWAP-70, LSM14A, and RDX) . High miR-378a (strand not specified) expression in renal carcinoma correlated with higher levels of endothelial surface marker CD34 in these tumors .
Notably, a recent study suggested clinical relevance for miR-378a in metastatic colorectal cancer, in which enhanced miR-378a expression significantly improved the sensitivity to cetuximab treatment in these patients .
Interestingly, recent data indicate a role of miR-378a in stem cells. miR-378a-5p transfection of MSCs has been shown to enhance their survival and angiogenic potential under hypoxic conditions in vitro . In coculture with human umbilical vein endothelial cells (HUVECs), miR-378a-5p-transfected MSCs formed a larger number of vascular branches on Matrigel. In the MSCs transfected with miR-378a-5p, the expression of Bcl-2-associated X protein (BAX), which is an important proapoptotic regulator, was decreased, leading to a better survival .
It still has to be determined if the proangiogenic effect of miR-378a in vivo is confined to tumor angiogenesis, or if this effect is also present in physiological angiogenesis and regenerative neovascularization. Interestingly, new findings in wound healing studies found a rather opposite conclusion. Recently, it was reported that anti-miR-378a-5p enhances wound healing process by upregulating integrin beta-3 and vimentin .
The role of the host gene of miR-378a on angiogenesis has also been studied. PGC-1β was reported to have opposite effects in ischemia-induced angiogenesis. It was reported that PGC-1β induces angiogenesis in skeletal muscle, enhancing the expression of VEGF both in vitro and in vivo after (transgenic) overexpression . Accordingly, it was also found that VEGFA is upregulated in C2C12 myoblast cell line with PGC-1β overexpression. However, after a PCR-based gene array of 84 known angiogenic factors and further RT-PCR of individual genes, they concluded that PGC-1β triggered an antiangiogenic program . After inducing hind limb ischemia in PGC-1β overexpressing mice, an impaired reperfusion was noticed when compared to wild type littermates .
7. miR-378a in Inflammation
The role of inflammation in angiogenesis is studied the most in the context of cancer (e.g., reviewed in [119, 120]) but is certainly not limited to this pathology. Both lymphoid (reviewed in [121, 122]) and myeloid (reviewed in ) derived inflammatory cells affect angiogenesis in a stimulating or inhibitory manner. The role of miR-378a in inflammatory cells was reported and its anti-inflammatory effect could be suggested.
NK cells exert potent cytotoxic effects when activated by type I IFN from the host once infected . miR-378a was found to be downregulated in activated NK cells and further proved to target granzyme B. Thus, IFN-α activation decreases miR-378a expression and in turn augments NK cell cytotoxicity . Accordingly, suppression of miR-378 targeting granzyme B in NK cells resulted in inhibition of Dengue virus replication in vivo .
Macrophages are known to play either inhibitory or stimulatory roles in angiogenesis (reviewed by ). miRNAs have been proposed to regulate activation and polarization of macrophages (reviewed by [127, 128]). In a study of Rückerl et al. miR-378a-3p was identified as a part of the IL-4-driven activation program of anti-inflammatory macrophages (M2) . miR-378a-3p was highly upregulated after stimulation with IL-4 of peritoneal exudate cells of mice injected with the parasite Brugia malayi compared to controls and infected IL-4-knockout mice. The study identified several targets for miR-378a-3p within the PI3 K/Akt signaling pathway, which are important for proliferation but only partially responsible for M2 phenotype . Another study found miR-378a (strand not specified) expression upregulated after stimulation with cytokines like, for example, TNF-α and IL-6 .
In line with its potential role in macrophages, miR-378a has been suggested as being of importance in the osteoclastogenesis . Mmu-miR-378a (strand not specified) has been found to be upregulated during osteoclastogenesis in vitro . Furthermore, serum levels of miR-378a-3p have been shown to correlate with bone metastasis burden in mice injected with mouse mammary tumor cell lines 4T1 and 4T1.2 .
A growing body of evidence suggests a role for miR-378a as a mediator controlling reciprocally dependent processes in metabolism, muscle differentiation/regeneration, and angiogenesis.
As miR-378a was found to be differentially regulated in different types of cancers and its level is changed in serum of prostate, renal, and gastric cancer patients, it can be considered as a biomarker for those diseases. The correlation between miR-378a expression and disease progression in lung cancer, liver cancer, and rhabdomyosarcoma suggests a further role of this microRNA as a prognostic marker.
Currently, miR-378a is not utilized as a therapeutic molecule. However, if more research will be done to the mechanisms of action, possibilities for therapeutic use of miR-378a could be sought in the field of metabolic disorders, obesity, or tumors. More studies on the effect of miR-378a expression in muscle disorders would also be desirable.
The proangiogenic effect of miR-378a was observed in tumors however, no studies have been performed on the angiogenic effects of miR-378a in physiological settings or diseases where angiogenesis plays important roles, such as diabetes and cardiovascular diseases. More study has to be done to assess the mechanisms of miR-378a function in blood vessel formation. Of note, in contrast with proangiogenic role of miR-378a, inhibition of miR-378a-5p enhanced wound healing process. This might suggest a role for miR-378a-5p in diseases such as diabetes or in decubitus ulcers, in which wound healing is impaired.
Of note is the confusion that has arises because of a disarray in nomenclature with studies describing the same molecule, miR-378a, as miR-422b, miR-378, or miR-37 . In addition, it is not always clear which of the two mature strands of miR-378a is studied. This could lead to misunderstandings and errors in interpreting the data published so far.
The graphical art (Figure 1) was performed with the use of Servier Medical Art.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by the MAESTRO (2012/06/A/NZ1/0004) and OPUS (2012/07/B/NZ1/0288) grants of the Polish National Science Centre and the Iuventus Plus (0244/IP1/2013/72) from the Ministry of Science and Higher Education. Faculty of Biochemistry, Biophysics and Biotechnology is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education.
Copyright © 2015 Bart Krist et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Kevin Gatter: Deceased: Kevin Gatter
Department of Oncology, University Hospital of North Norway, Tromso, Norway
Institute of Clinical Medicine, The Arctic University of Norway, Tromso, Norway
Tumour Biology Team, Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London, UK
Andrew R. Reynolds & Peter B. Vermeulen
Oncology Translational Medicine Unit, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, UK
Biological Sciences Platform, Sunnybrook Research Institute, Toronto, Canada
Elizabeth A. Kuczynski & Robert S. Kerbel
Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, UK
Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
Kevin Gatter & Francesco Pezzella
Translational Cancer Research Unit, GZA, Hospitals St Augustinus, University of Antwerp, Wilrijk-Antwerp, Belgium
HistoGeneX, Antwerp, Belgium
Department of Medical Biophysics, University of Toronto, Toronto, Canada
Oxford University Department of Oncology, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK
Hypoxia, the HIFs, Angiogenesis, and Vascular Diseases
Occlusive vascular diseases remain the most important causes of death and morbidity in industrialized societies. 109 Treatment of end stages such as myocardial infarction, peripheral artery disease (PAD), and stroke is usually limited only to palliative interventions, such as angioplasty and, in severe PAD cases, limb amputation. 110,111 The ability to induce and regulate angiogenesis and vascular remodeling in a directed manner would represent a major advance in the treatment of ischemic vascular diseases. Recent studies have demonstrated how hypoxia and the HIF pathway, through the modulation of angiogenic genes, such as Vegf ( Table 3 and Figure 3 ), regulate the adult vascular system in numerous pathological conditions. 88,112 -116 Not surprisingly, the HIF pathway represents an attractive therapeutic target in ischemic diseases, and its continued study in these contexts has revealed important insights ( Figure 4 ).
Hypoxia, the HIFs, and Peripheral Artery Disease
Peripheral artery disease is a condition characterized by obstruction of large arteries leading to vascular dysfunction in the extremities. 110,117 Vessel occlusion in PAD is usually caused by the development of atherosclerotic plaques, which are initiated by injuries to the endothelium from hemodynamic stress and uptake of oxidized low-density lipoprotein. Endothelial damage stimulates rapid proliferation of vascular smooth muscle cells, thickening the arterial wall. Nascent plaques are subsequently infiltrated by large numbers of macrophages, which drastically increase metabolic demand and cause local tissue hypoxia and induction of an angiogenic response. 118 Plaque perfusion promotes expansion by allowing more efficient infiltration of macrophages through new capillaries. 119 Interestingly, HIF-1α and VEGF are expressed in the plaque, suggesting that the HIF signaling pathway is directly involved in plaque angiogenesis. 120 Continued plaque growth progressively narrows existing arteries, restricting blood flow and reducing perfusion to adjacent tissues. Atherosclerotic stenosis (narrowing) of peripheral arteries leads to critical limb ischemia in 1% to 2% of patients. 121 The most severe form of PAD, critical limb ischemia, is characterized by pain at rest, ulceration, and/or gangrene. Although a neovascular response in the affected limb is mounted, it is often insufficient to adequately reperfuse the ischemic tissue, which eventually requires amputation. The HIFs promote the neoangiogenic response to tissue ischemia and pathophysiological angiogenesis in PAD, as levels of HIF-α mRNA, protein, and transcriptional targets increase following ischemic insult in animal models. 114,116 HIF transcriptional activity leads to the production of new capillaries via angiogenesis and the remodeling of existing arteries to accept increased flow, a process called arteriogenesis. 122 Surgical ligation of the femoral artery leads to a severe decrease in hindlimb blood flow, usually to 㰐% to 20% of the nonligated side, and serves as an animal model of PAD. 123 -125 Reduced blood flow in the main limb artery induces ischemia distally, which triggers a prototypical angiogenic and arteriogenic response. 126 The HIFs play an important role in the blood flow recovery during hindlimb ischemia, which is mainly achieved by arteriogenesis while angiogenesis plays a secondary role. 127,128 For example, femoral artery ligation (FAL) experiments revealed decreased limb perfusion and increased spontaneous amputation in Hif-1α +/– mice, which have a severely blunted induction of HIF-1α protein in response to hypoxia. 116 Administration of the HIF-1α inhibitor 2-methoxyestradiol phenocopied the defects observed in Hif-1α +/– mice. In contrast, forced expression of a constitutively active form of HIF-1α that is resistant to O2-dependent regulation, AdCA5, 112 stimulated reperfusion following FAL in both mouse and rabbit models. 114,129 AdCA5 treatment significantly increases the arteriogenic response, characterized by enlarged collateral blood vessels, demonstrating that HIF activity promotes vessel remodeling in addition to angiogenesis.
Recent work suggests several risk factors for PAD may mechanistically operate through the HIF pathway. As mice age, the ischemic accumulation of HIF-1α and its transcriptional targets diminishes following FAL. 116 Consequently, recovery from reperfusion is also impaired in old mice, which exhibit increased frequency of spontaneous limb amputation. Reduced HIF target gene expression in the limbs of old mice correlates with observed deficits in reperfusion. Diabetic mice also exhibit defective reperfusion following FAL and impaired wound healing. 130 When exposed to high glucose and low O2, dermal fibroblasts derived from Leprdb/db diabetic mice fail to stabilize HIF-1α protein and induce VEGF. 131,132 Expression of HIF-1α and VEGF levels is attenuated in diabetic mice subjected to FAL, suggesting defective HIF induction may underlie some of the vascular complications common to diabetics. 133,134 Mechanistically, diabetic mice exhibit defects in HIF signaling because high glucose impairs the activity of HIF-1α by inhibiting HIF-1α/Arnt heterodimerization and HIF-1α/p300 binding. Therapeutic enhancement of HIF activity can overcome age and diabetes, as ectopic expression of HIF-1α can partially rescue limb perfusion in old mice, 116 whereas activation of the HIF pathway via PHD inhibition reverses deficits in neovascularization observed in diabetic mice. 135 The importance of aging and diabetes as modulators of ischemic responses is becoming increasingly clear and will likely have significant functional importance for all ischemic diseases.
In addition to angiogenesis and arteriogenesis, the mobilization of circulating CACs appears to play an important role in the vascular response to ischemia. CACs are a heterogeneous cell population derived from the bone marrow that includes endothelial progenitor cells, circulating endothelial cells, hematopoietic progenitor cells, and mesenchymal stem cells. 136 -138 Stimulating cytokines such as HIF targets VEGF, placenta growth factor (PLGF), and SDF-1 induce CAC migration from the bone marrow and into the circulatory system. 139 CACs migrate to sites of ischemia where they promote vascular remodeling and stimulate angiogenesis and arteriogenesis. In models of ischemia-induced neovascularization, including FAL, tumors, wound healing, and choroidal neovascularization, CACs have been shown to incorporate into new blood vessels and promote neovascularization. 116,129,134,137,140 -145 SDF-1/CXCR4 and VEGF/VEGF-R2 signaling regulate the recruitment of these cells. 146 Ectopic expression of HIF-1α enhances the mobilization and recruitment of CACs to ischemic sites in the FAL model by upregulating SDF-1 and VEGF. 116 Consistently, diabetic mice also exhibit reduced CAC activity, which can be rescued by forced expression of HIF-1α or PHD inhibition via desferrioxamine (DFO) or dimethyloxalyglycine (DMOG). 133,135,147 HIF-1α has been heavily scrutinized in preclinical studies of PAD, but the role of HIF-2α is less clear.
Given that HIF-2α is highly expressed in the endothelium, it is likely to play a role in PAD. Indeed, studies in Phd1 knockout mice provide further evidence that HIF-2α can buffer tissues from hypoxic/ischemic stress. 148,149 When placed under ischemic stress, Phd1 –/– limb skeletal muscle is protected from oxidative damage and cell death. The ischemic tolerance in Phd1 –/– skeletal muscle is predominantly dependent on HIF-2α, as simultaneous deletion of both Phd1 and Hif-2α blocked the protective effects of PHD1 deficiency. HIF-2α may promote this tolerance by modulating glucose metabolism indirectly through regulation of PPARα, which is essential for ischemic tolerance in Phd1-deficient skeletal muscle. 148 PDK4, which limits mitochondrial glucose metabolism by inhibiting the pyruvate dehydrogenase complex, is a target of PPARα. 150 Alternatively, HIF-2α may contribute to the ischemic phenotypes in Phd1 –/– skeletal muscle by regulating redox homeostasis. 151,152 These studies demonstrate the diverse mechanisms through which hypoxia and the HIF pathway regulate vascular responses to tissue hypoxia and ischemia.
Hypoxia, the HIFs, and Ischemia-Induced Coronary Collateralization
In addition to its role in PAD, atherosclerosis also affects the coronary arteries, where atherosclerotic stenosis and luminal occlusion induce myocardial infarction and ischemia/hypoxia stimulates collateralization. Sixty percent of patients with coronary artery disease develop collateral vessels that bypass the stenosis. 153,154 The presence of collaterals is associated with reduced infarct size, less severe functional deterioration, and reduced mortality following myocardial infarction. 110,117,153,154 Patients who do not develop collateral vessels to increase blood flow distal to the site of stenosis are more likely to develop heart failure. 154 Recent studies indicate that deficits in the hypoxic response underlie phenotypic heterogeneity observed with respect to collateralization. For instance, monocytes isolated from patients with coronary artery disease (CAD) with collaterals produce greater amounts of VEGF in response to hypoxia compared to monocytes from patients without collaterals. 155 Moreover, increased HIF-1α expression in other blood cells (e.g., leukocytes) has also been associated with the presence of coronary collaterals in patients with CAD. Following this observation, the frequency of single-nucleotide polymorphisms (SNPs) in the human HIF1A gene was significantly higher in CAD without collaterals and also increased in patients with stable exertional angina over those presenting with myocardial infarction. 156,157 These HIF1A variants are less functional, as Hif-1α null mouse embryonic fibroblasts (MEFs) transfected with the HIF1A variants fail to upregulate HIF target genes as well as wild-type HIF1A. Defective HIF-1α activity, therefore, may lead to early onset symptoms. Genetic screening for less functional HIF-1α variants could aid early clinical evaluation of patients who are likely to develop advanced coronary disease.
Activation of HIFs in Ischemic Diseases as a Novel Therapy
Administration of angiogenic factors, including VEGF and FGF, and transplantation of bone marrow cells have been tested as potential treatments to enhance vascularization in ischemic diseases. However, increasing HIF-α levels and activity may represent a superior therapeutic approach due to the multiple pro-angiogenic pathways it regulates ( Figure 4 ). Blood vessels formed in pathological conditions are typically abnormal, tortuous, and leaky. These types of blood vessels are often observed experimentally when induced with a single agent, such as VEGF. To best treat ischemic diseases, normal vessels need to be formed. Interestingly, mice expressing constitutively active forms of HIF-1α and HIF-2α that are refractory to O2-dependent regulation are hypervascular, 158 -160 but in contrast to VEGF overexpressing mice, they possess “normalized” vessels. 159 In a tumor vessel abnormalization model, endothelial PHD2, via regulation of HIF-2α, senses and readapts O2 supply in response to O2 deprivation. 161 Haplodeficiency of Phd2 in ECs does not affect tumor vessel density and area, tortuosity, or lumen size but induces “normalization” of the endothelial lining, barrier, and stability. Interestingly, tumor vessels in Phd2 +/– mice are lined by a single-monolayer phalanx of regular, orderly formed, polarized cobblestone ECs, which have few fenestrations. These changes in EC shape, not numbers, do not affect primary tumor growth but improve tumor perfusion and oxygenation. 161 Inhibition of PHD2 in ECs would provide a conceptually different strategy to blocking angiogenesis, whereby tumor vessel function is improved by streamlining the EC layer and improving delivery of oxygen to tumors. This would render the tumors less malignant and metastatic, making them more vulnerable to radiation and chemotherapy.
Several strategies to promote HIF activity and angiogenesis are in development for use in ischemic diseases and have been employed in various preclinical models. Exposure to mild hypoxia, called hypoxic preconditioning, protects against ischemic challenge by inducing HIF-α accumulation. This approach can prevent apoptosis, which is seen in the retina where hypoxic preconditioning protects photoreceptors from light-induced damage. 162 Angiogenesis is also stimulated by hypoxic preconditioning, which has been documented in the myocardium. 163 The therapeutic utility of this intervention is limited, given that hypoxic treatment must precede the ischemic stress. Alternatively, ectopic expression of the HIF-α subunits represents an attractive option. Electroporation of plasmid DNA encoding HIF-1α enhanced the angiogenic response in models of hindlimb ischemia and wound healing. 116,133,135,164 Consistent with this finding, adenoviral delivery of an O2-refractory form of HIF-1α exhibited benefit in limb ischemia models in aged and diabetic mice. 116,129,165 A similar approach, treatment with DNA encoding the N-terminus of HIF-1α fused to the VP16 transactivation domain, also stimulated angiogenesis in animal models 166 -168 and is currently progressing through phase I and II clinical studies in patients with severe PAD. 169
These approaches, however, suffer from many of the challenges common to gene therapy and must be cautiously evaluated. An alternative approach to enhancing HIF signaling is to inhibit HIF-α degradation. Proteasomal inhibition with the macrophage-derived peptide PR39 inhibits HIF degradation and increases angiogenesis in ischemic mouse cardiac tissue. 170 As the PHD enzymes are the primary O2 sensing proteins, they have been pharmacologically targeted with the aim of augmenting HIF activity. As stated previously, PHD enzymes require O2, iron, and 2-oxoglutarate (2-OG) for their enzymatic activity. 16 Accordingly, iron chelators, such as DFO, exhibit potent HIF-α stabilizing activity both in vitro and in vivo. 17,171 2-OG analogues can also inhibit PHD activity by displacing the 2-OG cofactor in the PHD enzyme. DMOG is a cell-permeable precursor to the 2-OG analogue N-oxalylglycine that is effective both in vitro and in vivo. 16 PHD inhibitors are not specific to PHD1-3, as they likely inhibit all iron and 2-OG-dependent dioxygenases, of which 60 to 80 have been identified in humans. For example, lysyl hydroxylase, which is important in collagen biosynthesis, would also be affected. 172,173 Therefore, caution should be used when drawing conclusions based solely on PHD inhibitors, as the off-target effects of these compounds are not fully understood.
Hypoxia and HIFs in Blindness Disorders
Uncontrolled blood vessel growth is a central pathological component of many human blindness disorders, including diabetic retinopathy, age-related macular degeneration (AMD), glaucoma, and retinopathy of prematurity (ROP). Neuronal cell death and vision loss observed in these diseases are caused by aberrant, leaky vessels, which are often associated with pathological neovascularization. Collectively, these diseases are the most common cause of vision loss in America today, suggesting that highly effective anti-angiogenic therapy would have a tremendous effect on the prevalence of blindness in the industrialized world. 174 In fact, neovascular eye diseases are the clearest example of the therapeutic utility of blocking angiogenesis, as VEGF inhibitors have been successfully employed in the clinic and shown efficacy in treatment of the wet (neovascular) form of AMD. 175
Although strategies to inhibit VEGF have found success in the clinic, it is clear that other pro-angiogenic factors play an important role in this disease, as anti-VEGF therapies are ineffective in approximately 50% of patients. 175,176 Indeed, simultaneous blockade of multiple pro-angiogenic factors yielded significantly better results in mouse models of ocular neovascularization. 96 Given that the HIF pathway is a master regulator of angiogenesis and modulates multiple pro-angiogenic pathways, it is an attractive target for new therapeutic strategies ( Figure 3 ). Although the role of HIFs in neovascular eye disease has not been extensively evaluated, HIF-1α and HIF-2α are expressed in ECs and macrophages from neovascular membranes harvested from AMD patients. 177 Furthermore, HIF-1α and HIF-2α are both induced in a cell type–specific manner in a murine model of ROP, suggesting hypoxia plays a role in these diseases. 178 Indeed, the HIF pathway has a functional role in ROP, as systemic reduction of HIF-2α expression with a hypomorphic Hif-2α allele caused marked decreases in retinal neovascularization that was accompanied by defects in EPO expression. 179 Moreover, specific deletion of Hif-2α in astrocytes attenuated the neovascular response in the murine ROP model but did not affect developmental retinal angiogenesis. 180 The functional role of HIF-1α in pathological ocular neovascularization is less clear, although its integral role in other neovascular models strongly suggests it is an important regulator of retinal neovascularization. Indeed, the HIF inhibitor digoxin effectively suppressed neovascularization in mouse models of ROP and wet AMD. 181 Both models exhibited attenuated HIF-1α accumulation, VEGF induction, and recruitment of macrophages and bone marrowrived cells, suggesting HIF inhibition blocks many processes associated with ocular neovascularization. Although off-target effects of digoxin cannot be completely ruled out, these data provide a valuable proof of concept that HIF inhibition can effectively treat neovascular eye disease.
Therapeutic enhancement of HIF activity may also represent an important preventive therapy for retinopathy of prematurity, the second most common cause of blindness for children younger than 6 years old. ROP is a neovascular eye disease that affects premature infants. The retinal vasculature forms late in embryonic development and is thought to be regulated by retinal hypoxia. As the embryonic environment is relatively hypoxic, premature infants experience relative hyperoxia, which inhibits vascular growth and obliterates newly formed vessels of the eye. Continued retinal development increases the metabolic demands of the now hypoperfused retina and generates areas of hypoxia that promote a strong neovascular response. Retinal detachment, hemorrhage, and retinal damage are caused by neovascularization in ROP, leading to severe vision loss and blindness in some cases. Given the pathogenesis of this disease, it is not surprising that hypoxia plays a critical role in ROP. Pharmacologic activation of the HIF pathway by administration of the PHD inhibitor, DMOG, during the hyperoxic vaso-obliterative phase of ROP significantly abrogates vessel destruction and the resultant neovascularization in a mouse model. 182 Thus, therapeutic blockade and enhancement of HIF activity represent viable strategies for the treatment of many human blindness disorders ( Figure 4 ).
Types of Angiogenesis
There are two main types of angiogenesis (there are also less common types not discussed here):
It's important to note that when angiogenesis is triggered by hypoxia (as in cancer), the blood vessels that are produced aren't "normal" but rather structurally abnormal so that they are distributed unevenly in a tumor, and even then, blood flow can be uneven and inconsistent.
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New Angiogenesis Finding May Help Fight Cancer Growth
A researcher at the University of Wisconsin-Madison School of Medicine and Public Health has discovered a new part of the complicated mechanism that governs the formation of blood vessels, or angiogenesis.
The finding may help halt tumor growth in cancer patients, says Emery Bresnick, the senior author on the study, a professor of pharmacology and member of the UW-Madison Paul P. Carbone Comprehensive Cancer Center.
The research, published in the Journal of Cell Biology on Sept. 25, is the first to connect a particular nervous-system chemical to the regulation of blood vessels.
Normally, blood vessels form when wounds heal and during menstruation, pregnancy and fetal development. But impaired blood-vessel development and function are also a major cause of blindness, and tumors rely on new blood vessels as they develop.
Like most critical body processes, angiogenesis is tightly controlled by multiple balancing mechanisms. When Bresnick and colleagues, including postdoctoral fellow Soumen Paul, began the new study, they were not looking into angiogenesis. Instead, they were studying a protein that regulates the maturation of blood cells, and noticed that it turns on a gene that makes a compound called neurokinin-B, or NK-B.
Aware that NK-B affects cells in the nervous system, Bresnick wondered, "Why would a protein involved in blood-cell formation turn on the gene for a compound that is supposedly involved in regulating the nervous system?"
The researchers searched for NK-B receptors - molecules that can "recognize" and respond to NK-B - and found great numbers of them on endothelial cells, which line the inside of blood vessels.
Endothelial cells form the internal structure of a blood vessel, and during angiogenesis, they migrate, starting an extension of the blood-vessel network. When Paul added NK-B to endothelial cells, "They lost the capacity to organize in three dimensions, to form the tubes that are the precursors to new blood vessels," Bresnick says. "Then we got excited."
Further tests showed that NK-B could inhibit angiogenesis in four ways. It prevents the production of vascular endothelial growth factor (VEGF), a key stimulator of blood-vessel formation, and also reduces the number of receptor molecules that respond to VEGF. NK-B also slows the movement of endothelial cells, which is necessary to form new vessels, and raises the level of a newly discovered angiogenesis inhibitor.
"It's premature to call it a master switch, but intriguingly, it regulates at least four different processes, each of which individually would be anti-angiogenic," says Bresnick.
Angiogenesis inhibitors, Bresnick observes, are a fast-growing field of medicine. This June, the Food and Drug Administration approved an angiogenesis inhibitor as the first drug that can restore some vision in the more severe ("wet") form of age-related macular degeneration (AMD). Wet AMD occurs when leaky blood vessels form in the retina. Along with a similar growth of new blood vessels in diabetes, it is the major cause of blindness in older adults.
But the "holy grail" of angiogenesis inhibition concerns cancer treatment. Before solid tumors start to grow, they must create a new blood supply, and since adults need angiogenesis only during pregnancy and to heal wounds, blocking angiogenesis could be a promising way to halt tumor growth. Also in June, the FDA approved a compound that inhibits VEGF for treating colon cancer, the second-leading cause of cancer death in the United States. The VEGF-inhibitor reduces the formation of blood vessels, helping starve tumors.
But angiogenesis regulation is a two-way street, and there are some diseases in which it might be desirable to stimulate angiogenesis. The new research shows that the NK-B system can work both ways: Reducing inhibition seems to increase angiogenesis.
"Activating the NK-B receptor blocked angiogenesis, and blocking the receptor stimulated angiogenesis," Bresnick says. In theory, selectively stimulating angiogenesis could help treat heart attacks by restoring blood flow to the heart, increasing the blood supply to threatened heart muscle.
NK-B also plays a role in a mysterious but common syndrome called preeclampsia, in which soaring blood pressure and low blood oxygen levels harm or even kill pregnant women and their babies. Philip Lowry, at the University of Reading in the United Kingdom, has found that NK-B levels spike in preeclampsia, and the new understanding of NK-B's role in angiogenesis suggests that faulty blood-vessel formation may be to blame.
Because NK-B prevents endothelial cells from organizing into blood vessels, Bresnick says, "Maybe excess levels of NK-B are responsible for or contribute to impaired vascular development/function and certain symptoms of preeclampsia." According to the Preeclampsia Foundation, the condition affects about 200,000 American women each year.
Many angiogenesis inhibitors are under study at this point, but finding a regulatory molecule that affects four separate mechanisms "makes for an interesting package," Bresnick says.
The Wisconsin Alumni Research Foundation has applied for a patent on the discovery, which, says Bresnick, reflected the work of "outstanding collaborators at the University of Wisconsin-Madison, who facilitated this multidisciplinary study and co-authored this paper." Authors included Patricia Keely in the Department of Pharmacology John Fallon and Tim Gomez in the Department of Anatomy and Sam Gellman in the Department of Chemistry.
Bresnick and his collaborators are looking further into how the molecule works in human cells and in mouse models of angiogenesis.
Eventually, after years of basic research and drug development, the multitalented compound NK-B could wind up playing a major role in treating cancer and other diseases where blood vessel formation goes awry, Bresnick says. "We have discovered a new peptide that clearly suppresses angiogenesis via a novel multi-component mechanism," he says. "A key question is whether we can exploit it to develop therapeutics."
Application in Medicine
Angiogenesis as a Therapeutic Target
Angiogenesis may be a target for combating diseases characterized by either poor vascularisation or abnormal vasculature. Application of specific compounds that may inhibit or induce the creation of new blood vessels in the body may help combat such diseases.
The presence of blood vessels where there should be none may affect the mechanical properties of a tissue, increasing the likelihood of failure. The absence of blood vessels in a repairing or otherwise metabolically active tissue may inhibit repair or other essential functions.
Several diseases, such as ischemic chronic wounds, are the result of failure or insufficient blood vessel formation and may be treated by a local expansion of blood vessels, thus bringing new nutrients to the site, facilitating repair. Other diseases, such as age-related macular degeneration, may be created by a local expansion of blood vessels, interfering with normal physiological processes.
The modern clinical application of the principle of angiogenesis can be divided into two main areas: anti-angiogenic therapies, which angiogenic research began with, and pro-angiogenic therapies. Whereas anti-angiogenic therapies are being employed to fight cancer and malignancies, which require an abundance of oxygen and nutrients to proliferate, pro-angiogenic therapies are being explored as options to treat cardiovascular diseases, the number one cause of death in the Western world.
One of the first applications of pro-angiogenic methods in humans was a German trial using fibroblast growth factor 1 (FGF-1) for the treatment of coronary artery disease. Clinical research in therapeutic angiogenesis is ongoing for a variety of atherosclerotic diseases, like coronary heart disease, peripheral arterial disease, wound healing disorders, etc.
Also, regarding the mechanism of action, pro-angiogenic methods can be differentiated into three main categories: gene-therapy, targeting genes of interest for amplification or inhibition protein-therapy, which primarily manipulates angiogenic growth factors like FGF-1 or vascular endothelial growth factor, VEGF and cell-based therapies, which involve the implantation of specific cell types.
There are still serious, unsolved problems related to gene therapy. Difficulties include effective integration of the therapeutic genes into the genome of target cells, reducing the risk of an undesired immune response, potential toxicity, immunogenicity, inflammatory responses, and oncogenesis related to the viral vectors used in implanting genes and the sheer complexity of the genetic basis of angiogenesis.
The most commonly occurring disorders in humans, such as heart disease, high blood pressure, diabetes and Alzheimer&rsquos disease, are most likely caused by the combined effects of variations in many genes, and, thus, injecting a single gene may not be significantly beneficial in such diseases.
Oral, intravenous, intra-arterial, or intramuscular routes of protein administration are not always as effective, as the therapeutic protein may be metabolized or cleared before it can enter the target tissue. Cell-based pro-angiogenic therapies are still early stages of research, with many open questions regarding best cell types and dosages to use.
Cancer cells are cells that have lost their ability to divide in a controlled fashion. A malignant tumor consists of a population of rapidly dividing and growing cancer cells that progressively accrues mutations. However, tumors need a dedicated blood supply to provide the oxygen and other essential nutrients they require in order to grow beyond a certain size (generally 1–2 mm3).
Tumors induce blood vessel growth (angiogenesis) by secreting various growth factors (e.g. VEGF). Growth factors such as bFGF and VEGF can induce capillary growth into the tumor, which some researchers suspect supply required nutrients, allowing for tumor expansion.
Unlike normal blood vessels, tumor blood vessels are dilated with an irregular shape. In 2007, it was discovered that cancerous cells stop producing the anti-VEGF enzyme PKG. In normal cells (but not in cancerous ones), PKG apparently limits beta-catenin, which solicits angiogenesis.
Other clinicians believe angiogenesis really serves as a waste pathway, taking away the biological end products secreted by rapidly dividing cancer cells. In either case, angiogenesis is a necessary and required step for transition from a small harmless cluster of cells, often said to be about the size of the metal ball at the end of a ball-point pen, to a large tumor.
Angiogenesis is also required for the spread of a tumor, or metastasis. Single cancer cells can break away from an established solid tumor, enter the blood vessel, and be carried to a distant site, where they can implant and begin the growth of a secondary tumor.
Evidence now suggests the blood vessel in a given solid tumor may, in fact, be mosaic vessels, composed of endothelial cells and tumor cells. This mosaicity allows for substantial shedding of tumor cells into the vasculature, possibly contributing to the appearance of circulating tumor cells in the peripheral blood of patients with malignancies. The subsequent growth of such metastases will also require a supply of nutrients and oxygen and a waste disposal pathway.
Endothelial cells have long been considered genetically more stable than cancer cells. This genomic stability confers an advantage to targeting endothelial cells using antiangiogenic therapy, compared to chemotherapy directed at cancer cells, which rapidly mutate and acquire &lsquodrug resistance&rsquo to treatment. For this reason, endothelial cells are thought to be an ideal target for therapies directed against them.
Recent studies by Klagsbrun, et al. have shown, however, that endothelial cells growing within tumors do carry genetic abnormalities. Thus, tumor vessels have the theoretical potential for developing acquired resistance to drugs. This is a new area of angiogenesis research being actively pursued.
Two independent studies published in the journal Nature in 2010 November confirmed the ability of tumors to make their own blood vessels. When one group found that tumor stem cells could make their own blood vessels and avastin could not inhibit their early differentiation, the other group showed that selective targeting of endothelial cells generated by tumor-derived stem cells in mouse xenografts resulted in tumour reduction. These studies done in glioblastoma model may have implications in other tumors.
To Learn More:
The Angiogenesis Foundation http://www.angio.org/ Tumor Angiogenesis Regulators Ruben R. Gonzalez-Perez, Bo R. Rueda CRC Press Angiogenesis Protocols (Methods in Molecular Biology) Stewart Martin, Cliff Murray Humana Press Visualizing Angiogenesis with GFP History of Research on Tumor Angiogenesis Domenico Ribatti Springer William Li: Can we eat to starve cancer? TEDTalk on anti-angiogenesis
Illustration: Confocal micrograph of new blood vessel formation. Credit Denise Stenzel, LRI, CRUK, Wellcome Images