Adipose Stromal Cells Amplify Angiogenic Signaling via the VEGF/mTOR/Akt Pathway in a Murine Hindlimb Ischemia Model: A 3D Multimodality Imaging Study

Although adipose-derived stromal cell (ADSC) transplantation has been demonstrated as a promising therapeutic strategy for peripheral arterial disease (PAD), the mechanism of action behind the observed therapeutic efficacy of ADSCs remains unclear. This study was designed to investigate the long-term outcome and therapeutic behavior of engrafted ADSCs in a murine hindlimb ischemia model using multimodality molecular imaging approaches. ADSCs (1.0×10⁷) were isolated from Tg(Fluc-egfp) mice which constitutively express dual-reporter firefly luciferase and enhanced green fluorescent protein (Fluc⁺-eGFP⁺, mADSCs^Fluc+GFP+), then intramuscularly injected into the hindlimb of BALB/c-nu mice after unilateral femoral artery ligation and excision. Abbreviated survival (∼5 weeks) of post-transplant mADSCs within the ischemic hindlimb was longitudinally monitored using noninvasive bioluminescence imaging (BLI), fluorescence imaging (FRI), and bioluminescence tomography with micro-computed tomography (BLT/micro-CT). Use of the BLT/micro-CT system enabled quantitative 3-dimensional (3D) imaging of the cells’ distribution and kinetics in vivo. Engrafted mADSCs improved blood perfusion recovery, ambulatory performance and prognosis of the ischemic hindlimb, probably by inducing angiogenesis and formation of collateral vessels, which could be visualized using laser Doppler perfusion imaging (LDPI), micro-CT angiography, vascular-cast imaging, and immunofluorescence. mADSCs augmented activation of the pro-angiogenic VEGF/mTOR/Akt pathway in vivo, even though the cells failed to incorporate into the host microvasculature as functional components. Downregulation of VEGF/mTOR/Akt signaling using small molecule inhibitors counteracted mADSC-induced angiogenesis and perfusion restoration. This study demonstrates for the first time the spatiotemporal kinetics and functional survival of transplanted mADSCs in a PAD model using in vivo 3D multimodality imaging. Our study indicates that mADSCs potentiate pro-angiogenic signal amplification via a VEGF/mTOR/Akt-dependent pathway, and thereby promote recovery from hindlimb ischemia.     

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.     

Therapeutic angiogenesis in a murine model of limb ischemia by recombinant periostin and its fasciclin I domain

Periostin, an extracellular matrix protein, is expressed in injured tissues, such as the heart with myocardial infarction, and promotes angiogenesis and tissue repair. However, the molecular mechanism associated with periostin-stimulated angiogenesis and tissue repair is still unclear. In order to clarify the role of periostin in neovascularization, we examined the effect of periostin in angiogenic potentials of human endothelial colony forming cells (ECFCs) in vitro and in an ischemic limb animal model. Recombinant periostin protein stimulated the migration and tube formation of ECFCs. To identify the functional domains of periostin implicated in angiogenesis, five fragments of periostin, including four repeating FAS-1 domains and a carboxyl terminal domain, were expressed in Escherichia coli and purified to homogeneity. Of the five different domains, the first FAS-1 domain stimulated the migration and tube formation of human ECFCs as potent as the whole periostin. Chemotactic migration of ECFCs induced by the full length and the first FAS-1 domain of periostin was abrogated by blocking antibodies against β3 and β5 integrins. Intramuscular injection of the full length and the first FAS-1 domain of periostin into the ischemic hindlimb of mice attenuated severe limb loss and promoted blood perfusion and homing of intravenously administered ECFCs to the ischemic limb. These results suggest that the first FAS-1 domain is responsible for periostin-induced migration and angiogenesis and it can be used as a therapeutic tool for treatment of peripheral artery occlusive disease by stimulating homing of ECFCs.     

Fibroblasts Derived from Human Pluripotent Stem Cells Activate Angiogenic Responses In Vitro and In Vivo

Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.     

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.     

Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood.     

Autophagic degradation of HAS2 in endothelial cells: A novel mechanism to regulate angiogenesis

Hyaluronan plays a key role in regulating inflammation and tumor angiogenesis. Of the three transmembrane hyaluronan synthases, HAS2 is the main pro-angiogenic enzyme responsible for excessive hyaluronan production. We discovered that HAS2 was degraded in vascular endothelial cells via autophagy evoked by nutrient deprivation, mTOR inhibition, or pro-autophagic proteoglycan fragments endorepellin and endostatin. Using live-cell and super-resolution confocal microscopy, we found that protracted autophagy evoked a dynamic interaction between HAS2 and ATG9A, a key transmembrane autophagic protein. This regulatory axis of HAS2 degradation occurred in various cell types and species and in vivo upon nutrient deprivation. Inhibiting in vivo autophagic flux via chloroquine showed increased levels of HAS2 in the heart and aorta. Functionally, autophagic induction via endorepellin or mTOR inhibition markedly suppressed extracellular hyaluronan production in vascular endothelial cells and inhibited ex vivo angiogenic sprouting. Thus, we propose autophagy as a novel catabolic mechanism regulating hyaluronan production in endothelial cells and demonstrate a new link between autophagy and angiogenesis that could lead to potential therapeutic modalities for angiogenesis.     

Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis

Mesenchymal stem cells (MSCs) accelerate regeneration of ischemic or injured tissues by stimulation of angiogenesis through a paracrine mechanism. Tumor necrosis factor-α (TNF-α)-activated MSCs secrete pro-angiogenic cytokines, including IL-6 and IL-8. In the present study, using an ischemic hindlimb animal model, we explored the role of IL-6 and IL-8 in the paracrine stimulation of angiogenesis and tissue regeneration by TNF-α-activated MSCs. Intramuscular injection of conditioned medium derived from TNF-α-treated MSCs (TNF-α CM) into the ischemic hindlimb resulted in attenuated severe limb loss and stimulated blood perfusion and angiogenesis in the ischemic limb. Immunodepletion of IL-6 and IL-8 resulted in attenuated TNF-α CM-stimulated tissue repair, blood perfusion, and angiogenesis. In addition, TNF-α CM induced migration of human cord blood-derived endothelial progenitor cells (EPCs) through IL-6- and IL-8-dependent mechanisms in vitro. Intramuscular injection of TNF-α CM into the ischemic limb led to augmented homing of tail vein-injected EPCs into the ischemic limb in vivo and immunodepletion of IL-6 or IL-8 from TNF-α CM attenuated TNF-α CM-stimulated homing of EPCs. In addition, intramuscular injection of recombinant IL-6 and IL-8 proteins resulted in increased homing of intravenously transplanted EPCs into the ischemic limb and improved blood perfusion in vivo. These results suggest that TNF-α CM stimulates angiogenesis and tissue repair through an increase in homing of EPCs through paracrine mechanisms involving IL-6 and IL-8.     

Therapeutic myocardial angiogenesis with vascular endothelial growth factors

Emerging evidence has shown that administration of angiogenic growth factors, either as recombinant protein or by gene transfer, can augment tissue perfusion through neovascularization in animal models of myocardial and hindlimb ischemia. Many cytokines have angiogenic activity; one of those that have been best studied in animal models and clinical trials is vascular endothelial growth factor (VEGF). VEGF has been known to be a key regulator of physiologic and pathologic angiogenesis associated with tumor. Recently the effect of VEGF is not restricted to the direct angiogenic effect in vivo but includes mobilization of bone-marrow-derived endothelial progenitor cells and augmentation of postnatal vasculogenesis in situ. Clinical trials of therapeutic angiogenesis with VEGF in patients with end-stage coronary artery disease have shown increases in exercise time and reductions in anginal symptoms and have provided objective evidence of improved perfusion and left ventricular function. Larger scale placebo-controlled trials with recombinant protein (rhVEGF₁₆₅) have been limited to intracoronary and intravenous administration and have shown favorable trends in exercise time and angina frequency. Small-scale, placebo-controlled, randomized clinical trials of gene transfer (phVEGF-2) via thoracotomy or percutaneous intramyocardial delivery demonstrated significant improvement of both subjective symptoms and objective measures of myocardial ischemia. Both therapeutic modalities appear to be safe and well tolerated. Further studies are required to determine the optimal dose, formulation, route of administration, and combinations of growth factors and the utility of adjunctive endothelial progenitor cell or other stem cell supplementation, to provide safe and effective therapeutic myocardial neovascularization. (Mol Cell Biochem 264: 63–74, 2004)     

Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells

We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.     

Tie2-Dependent Neovascularization of the Ischemic Hindlimb Is Mediated by Angiopoietin-2

The angiopoietins (ANGPT) are ligands for the endothelial cell (EC) receptor tyrosine kinase, Tie2. Angpt-1 is a Tie2 agonist that promotes vascular maturation and stabilization, whereas Angpt-2 is a partial agonist/antagonist involved in the initiation of postnatal angiogenesis. Therefore, we hypothesized that overexpression of Angpt-2 would be more effective than Angpt-1 for enhancing the perfusion recovery in the ischemic hindlimb. Perfusion recovery was markedly impaired in Tie2-deficient animals at day 35 in a model of chronic hindlimb ischemia. Injections of Angpt-2 or VEGFA plasmid at 7 days post femoral artery resection enhanced recovery and improved arteriogenesis as assessed by angiographic scores, whereas Angpt-1 or null plasmid had no effect. In addition, Angpt-2 together with VEGF resulted in greater improvement in perfusion and collateral vessel formation than VEGF alone. Similarly, conditional overexpression of Angpt-2 in mice improved ischemic limb blood flow recovery, while Angpt-1 overexpression was ineffective. These data from Tie2 heterozygote deficient mice demonstrate, for the first time, the importance of the Tie2 pathway in spontaneous neovascularization in response to chronic hindlimb ischemia. Moreover, they show that overexpression of the partial agonist, Angpt-2, but not Angpt-1, enhanced ischemic hind limb perfusion recovery and collateralization, suggesting that a coordinated sequence antagonist and agonist activity is required for effective therapeutic revascularization.     

Protein profiling and angiogenic effect of hypoxia-cultured human umbilical cord blood-derived mesenchymal stem cells in hindlimb ischemia

The aim of the present study was to investigate protein profiles of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) cultured in normoxic (21% O₂) and hypoxic (1% O₂) conditions, and evaluate oxygenation effects on angiogenesis in an ischemic hindlimb mouse model using a modified ischemic scoring system. Hypoxic conditions did not change the expression of phenotypic markers and increased adipogenesis and chondrogenesis. Epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), TGF-β RII, and vascular endothelial growth factor (VEGF) were upregulated in the conditioned medium of hypoxic hUCB-MSCs, which are commonly related to angiogenesis and proliferation of biological processes by Gene Ontology. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, significant enrichment of the phosphorylation of abelson murine leukemia viral oncogene homolog 1 (ABL1) (Phospho-Tyr204) and B-cell lymphoma-extra large (BCL-XL) (Phospho-Thr47) as anti-apoptotic pathways was observed in hypoxic hUCB-MSCs. Furthermore, hypoxic conditions induced proliferation and migration, and reduced apoptosis of hUCB-MSCs in vitro. Based on the results of protein antibody array, we evaluated the angiogenic effects of injecting normoxic or hypoxic hUCB-MSCs (1 × 10⁶) into the ischemic hindlimb muscles of mice. Ischemic scores and capillary generation were significantly greater in the hypoxic hUCB-MSC injection group than in the normoxic hUCB-MSC group. Our findings demonstrate that culturing hUCB-MSCs in hypoxic conditions not only significantly enriches phosphorylation in the anti-apoptosis pathway and enhances the secretion of several angiogenic proteins from cells, but also alleviates ischemic injury of hindlimb of mice.     

Lipocalin-7 is a matricellular regulator of angiogenesis

Background

Matricellular proteins are extracellular regulators of cellular adhesion, signaling and performing a variety of physiological behaviors such as proliferation, migration and differentiation. Within vascular microenvironments, matricellular proteins exert both positive and negative regulatory cues to vascular endothelium. The relative balance of these matricellular cues is believed to be critical for vascular homeostasis, angiogenesis activation or angiogenesis resolution. However, our knowledge of matricellular proteins within vascular microenvironments and the mechanisms by which these proteins impact vascular function remain largely undefined. The matricellular protein lipocalin-7 (LCN7) is found throughout vascular microenvironments, and circumstantial evidence suggests that LCN7 may be an important regulator of angiogenesis. Therefore, we hypothesized that LCN7 may be an important regulator of vascular function.

Methodology and Principal Findings

To test this hypothesis, we examined the effect of LCN7 overexpression, recombinant protein and gene knockdown in a series of in vitro and in vivo models of angiogenesis. We found that overexpression of LCN7 in MB114 and SVEC murine endothelial cell lines or administration of highly purified recombinant LCN7 protein increased endothelial cell invasion. Similarly, LCN7 increased angiogenic sprouting from quiescent endothelial cell monolayers and ex vivo aortic rings. Moreover, LCN7 increased endothelial cell sensitivity to TGF-β but did not affect sensitivity to other pro-angiogenic growth factors including bFGF and VEGF. Finally, morpholino based knockdown of LCN7 in zebrafish embryos specifically inhibited angiogenic sprouting but did not affect vasculogenesis within injected embryos.

Conclusions and Significance

No functional analysis has previously been performed to elucidate the function of LCN7 in vascular or other cellular processes. Collectively, our results show for the first time that LCN7 is an important pro-angiogenic matricellular protein of vascular microenvironments.     

Potential Compensation among Group I PAK Members in Hindlimb Ischemia and Wound Healing

PAKs are serine/threonine kinases that regulate cytoskeletal dynamics and cell migration. PAK1 is activated by binding to the small EF hand protein, CIB1, or to the Rho GTPases Rac1 or Cdc42. The role of PAK1 in angiogenesis was established based only on in vitro studies and its role in angiogenesis in vivo has never been examined. Here we tested the hypothesis that PAK1 is an essential regulator of ischemic neovascularization (arteriogenesis and angiogenesis) and wound healing using a global PAK1 knockout mouse. Neovascularization was assessed using unilateral hindlimb ischemia. We found that plantar perfusion, limb use and appearance were not significantly different between 6–8 week old PAK1^−/− and PAK1^+/+ mice throughout the 21-day period following hindlimb ischemia; however a slightly delayed healing was observed in 16 week old PAK1^−/− mice. In addition, the wound healing rate, as assessed with an ear punch assay, was unchanged in PAK1^−/− mice. Surprisingly, however, we observed a notable increase in PAK2 expression and phosphorylation in ischemic gastrocnemius tissue from PAK1^−/− but not PAK1^+/+ mice. Furthermore, we observed higher levels of activated ERK2, but not AKT, in ischemic and non-ischemic muscle of PAK1^−/− mice upon hindlimb ischemic injury. A group I PAK inhibitor, IPA3, significantly inhibited endothelial cell sprouting from aortic rings in both PAK1^−/− and PAK1^+/+ mice, implying that PAK2 is a potential contributor to this process. Taken together, our data indicate that while PAK1 has the potential to contribute to neovascularization and wound healing, PAK2 may functionally compensate when PAK1 is deficient.     

Angiogenic activity of sesamin through the activation of multiple signal pathways

The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125^FAK-, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.     

Reduced expression of let‐7f activates TGF‐β/ALK5 pathway and leads to impaired ischaemia‐induced neovascularization after cigarette smoke exposure

This study sought to determine the potential role of microRNAs (miRNAs) in the detrimental effects of cigarette smoke on angiogenesis and neovascularization. Using large‐scale miRNA profiling and qRT‐PCR analyses, we identified let‐7f as a pro‐angiogenic miRNA which expression is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischaemia, intramuscular injection of let‐7f mimic restored ischaemia‐induced neovascularization in MES. Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let‐7f mimic. Clinically, this was associated with reduced ambulatory impairment and hindlimb ischaemic damage. Treatment with let‐7f mimic could also rescue pro‐angiogenic cell (PAC) number and function (attachment, proliferation, migration) in MES. ALK5 (TGF‐βR1), an important modulator of angiogenesis, is a target of let‐7f. Here we show that ALK5 is increased in HUVECs exposed to CSE and in the ischaemic muscles of MES. This is associated with a downstream activation of the anti‐angiogenic factors SMAD2/3 and PAI‐1. Importantly, treatment with let‐7f mimic reduces the expression of ALK5, SMAD2/3 and PAI‐1 both in vitro and in vivo. Moreover, let‐7f overexpression or ALK5 inhibition can rescue angiogenesis in HUVECs exposed to CSE. Cigarette smoke exposure is associated with reduced expression of let‐7f and activation of the anti‐angiogenic TGF‐β/ALK5 pathway. Overexpression of let‐7f using a miRNA mimic could constitute a novel therapeutic strategy to improve ischaemia‐induced neovascularization in pathological conditions.     

Genetic heterogeneity of skin microvasculature

Angiogenesis, the formation of new blood vessels from existing vasculature, is a complex process that is essential for normal embryonic development. Current models for experimental evaluation of angiogenesis often use tissue from large vessels like the aorta and umbilical vein, which are phenotypically distinct from microvasculature. We demonstrate that the utilization of skin to measure microvascular angiogenesis in embryonic and adult tissues is an efficient way to quantify microvasculature angiogenesis. We validate this approach and demonstrate its added value by showing significant differences in angiogenesis in monogenic and polygenic mouse models. We discovered that the pattern of angiogenic response among inbred mouse strains in this ex vivo assay differs from the strain distributions of previous in vivo angiogenesis assays. The difference between the ex vivo and in vivo assays may be related to systemic factors present in whole animals. Expression analysis of cultured skin biopsies from strains of mice with opposing angiogenic response was performed to identify pathways that contribute to differential angiogenic response. Increased expression of negative regulators of angiogenesis in C57Bl/6J mice was associated with lower growth rates.