Angiogenesis In Ischemia Research Articles

Nitrite Anion Therapy Protects Against Chronic Ischemic Tissue Injury in db/db Diabetic Mice in a NO/VEGF-Dependent Manner

Nitrite anion has been demonstrated to be a prodrug of nitric oxide (NO) with positive effects on tissue ischemia/reperfusion injury, cytoprotection, and vasodilation. However, effects of nitrite anion therapy for ischemic tissue vascular remodeling during diabetes remain unknown. We examined whether sodium nitrite therapy altered ischemic revascularization in BKS-Leprdb/db mice subjected to permanent unilateral femoral artery ligation. Sodium nitrite therapy completely restored ischemic hind limb blood flow compared with nitrate or PBS therapy. Importantly, delayed nitrite therapy 5 days after ischemia restored ischemic limb blood flow in aged diabetic mice. Restoration of blood flow was associated with increases in ischemic tissue angiogenesis activity and cell proliferation. Moreover, nitrite but not nitrate therapy significantly prevented ischemia-mediated tissue necrosis in aged mice. Nitrite therapy significantly increased ischemic tissue vascular endothelial growth factor (VEGF) protein expression that was essential for nitrite-mediated reperfusion of ischemic hind limbs. Nitrite significantly increased ischemic tissue NO bioavailability along with concomitant reduction of superoxide formation. Lastly, nitrite treatment also significantly stimulated hypoxic endothelial cell proliferation and migration in the presence of high glucose in an NO/VEGF-dependent manner. These results demonstrate that nitrite therapy effectively stimulates ischemic tissue vascular remodeling in the setting of metabolic dysfunction that may be clinically useful.

Ketanserin improves cardiac performance after myocardial infarction in spontaneously hypertensive rats partially through restoration of baroreflex function

Baroreflex dysfunction is associated with a higher rate of sudden death after myocardial infarction (MI). Ketanserin enhances baroreflex function in rats. The present work was designed to examine whether ketanserin improves the post-MI cardiac function and to explore the possible mechanism involved. Spontaneously hypertensive rats (SHR) were treated with ketanserin (0.3 mg·kg(-1)·d(-1)). Two weeks later, blood pressure and baroreflex function were measured, followed by a ligation of the left coronary artery. The expressions of vesicular acetylcholine transporter (VAChT) and α7 nicotinic acetylcholine receptor (α7-nAChR) in ischemic myocardium, angiogenesis, cardiac function, and left ventricular (LV) remodeling were evaluated subsequently. Ketanserin significantly improved baroreflex sensitivity (0.62±0.21 vs 0.34±0.12 ms/mmHg, P<0.01) and vagal tonic activity (heart rate changes in response to atropine, 54.8±16.2 vs 37.6±13.4 bpm, P<0.01) without affecting the blood pressure or basic heart rate in SHR. Treatment of SHR with ketanserin prominently improved cardiac function and alleviated LV remodeling, as reflected by increases in the ejection fraction, fractional shortening, and LV systolic pressure as well as decreases in LV internal diameter and LV relative weight. The capillary density, vascular endothelial growth factor expression, and blood flow in the ischemic myocardium were significantly higher in the ketanserin-treated group. In addition, ketanserin markedly increased the expression of VAChT and α7-nAChR in ischemic myocardium. Ketanserin improved post-MI cardiac function and angiogenesis in ischemic myocardium. The findings provide a mechanistic basis for restoring baroreflex function using ketanserin in the treatment of MI.

Control of angiogenesis dictated by picomolar superoxide levels

Control of vascular insufficiencies due to various cardiovascular pathologies is important for developing specific and effective treatments. Fluctuations in oxidative stress significantly alter the progression of angiogenesis under physiological and pathological conditions. However, the precise amount of reactive oxygen species (ROS) required to influence subsequent signaling pathways for ischemic angiogenesis remains undefined. Here, we have determined the effect of ROS-mediated molecular mechanisms on angiogenesis in a murine model of peripheral artery disease using Gclm mutant mice (a model of compromised glutathione synthesis and therefore reduced antioxidant capacity). Left femoral artery ligation and excision were performed in Gclm WT (+/+), heterozygous (+/−), and null (−/−) mice. Blood flow (laser Doppler), angiogenic index (CD31/DAPI), and proliferation index (Ki67/DAPI) were significantly increased in Gclm+/− mice but not in Gclm+/+ or Gclm−/− mice. Measurements of reactive oxygen species suggest that the amount of superoxide required to stimulate angiogenesis after the induction of ischemia is 9.82pmol/mg of tissue. Protein carbonyl levels increased in a manner consistent with increasing oxidative stress. Superoxide and protein carbonyl levels were reduced by the addition of the nitroxide tempol, a known superoxide dismutase mimetic. Finally, restoration of blood flow in Gclm+/− mice was attenuated by a VEGF164 aptamer, verifying that slightly elevated levels of ROS restore blood flow by stimulating endothelial cell proliferation through a VEGF-dependent pathway. The results of this study reveal new information on the amount of ROS necessary for angiogenic activity and provide the foundation of critical redox parameters for vascular remodeling responses. The information obtained from this study on vascular ischemia, using a model of decreased antioxidant capacity, has provided insight into the control of revascularization and is a step forward in our ability to regulate angiogenic therapies.

Contrasting roles of E2F2 and E2F3 in endothelial cell growth and ischemic angiogenesis

The growth of new blood vessels after ischemic injury requires endothelial cells (ECs) to divide and proliferate, and the E2F transcription factors are key regulators of the genes responsible for cell-cycle progression; however, the specific roles of individual E2Fs in ECs are largely unknown. To determine the roles of E2F2 and E2F3 in EC proliferation and the angiogenic response to ischemic injury, hind–limb ischemia was surgically induced in E2F2−/− mice, endothelial-specific E2F3-knockout (EndoE2F3∆/∆) mice, and their littermates with wild-type E2F2 and E2F3 expression. Two weeks later, Laser-Doppler perfusion measurements, capillary density, and endothelial proliferation were significantly greater in E2F2−/− mice and significantly lower in EndoE2F3∆/∆ mice than in their littermates, and EndoE2F3∆/∆ mice also developed toe and limb necrosis. The loss of E2F2 expression was associated with increases in the proliferation and G1/S-phase gene expression of isolated ECs, while the loss of E2F3 expression led to declines in these parameters. Thus E2F2 impairs, and endothelial E2F3 promotes, the angiogenic response to peripheral ischemic injury through corresponding changes in EC cell-cycle progression.

Platelet neuropeptide Y is critical for ischemic revascularization in mice

We previously reported that the sympathetic neurotransmitter neuropeptide Y (NPY) is potently angiogenic, primarily through its Y2 receptor, and that endogenous NPY is crucial for capillary angiogenesis in rodent hindlimb ischemia. Here we sought to identify the source of NPY responsible for revascularization and its mechanisms of action. At d 3, NPY(-/-) mice demonstrated delayed recovery of blood flow and limb function, consistent with impaired collateral conductance, while ischemic capillary angiogenesis was reduced (~70%) at d 14. This biphasic temporal response was confirmed by 2 peaks of NPY activation in rats: a transient early increase in neuronally derived plasma NPY and increase in platelet NPY during late-phase recovery. Compared to NPY-null platelets, collagen-activated NPY-rich platelets were more mitogenic (~2-fold vs. ~1.6-fold increase) for human microvascular endothelial cells, and Y2/Y5 receptor antagonists ablated this difference in proliferation. In NPY(+/+) mice, ischemic angiogenesis was prevented by platelet depletion and then restored by transfusion of platelets from NPY(+/+) mice, but not NPY(-/-) mice. In thrombocytopenic NPY(-/-) mice, transfusion of wild-type platelets fully restored ischemia-induced angiogenesis. These findings suggest that neuronally derived NPY accelerates the early response to femoral artery ligation by promoting collateral conductance, while platelet-derived NPY is critical for sustained capillary angiogenesis.

Mesenchymal stem cells modified with miR-126 release angiogenic factors and activate Notch ligand Delta-like-4, enhancing ischemic angiogenesis and cell survival

The endothelial cell-specific microRNA (miRNA), miR-126, is considered a master regulator of physiological angiogenesis. Transplanted mesenchymal stem cells (MSCs) release soluble factors contributing to neoangiogenesis and cardiac repair. Therefore, we hypothesized that the over-expression of miR-126 may prolong MSC survival and enhance the cell secretome, thereby improving post-infarction angiogenesis and cardiac function. In this study, MSCs harvested from male C57BL/6 mouse bone marrow were infected in vitro with miR-126 (MSC(miR-126)) by using recombinant lentiviral vectors; the control cells were either non-transfected or transduced with mock vectors (MSC(null)). The results showed an increased secretion of angiogenic factors and a higher resistance against hypoxia in MSC(miR-126) compared with the control cells. The expression of the Notch ligand Delta-like (Dll)-4 in the MSC(miR-126) group was also increased. For in vivo experiments, MSC(miR-126) cultures were intramyocardially injected into the infarct region of the hearts of female C57BL/6 mice (an acute myocardial infarction model) who had undergone ligation of the left anterior descending coronary artery. The survival of MSC(miR-126) cultures, determined by Sry expression, was increased at 7 days after transplantation. MSC(miR-126)-treated animals showed significantly improved cardiac function as assessed by echocardiography 2 weeks later. The expression levels of angiogenic factors and Dll-4 in the infarcted myocardium were further increased by MSC(miR-126) compared with MSCs or MSC(null) cultures. Furthermore, fluorescent microsphere and histological studies revealed that myocardial blood flow and microvessel density were significantly increased in the MSC(miR-126)-transplanted animals. In addition, we found increased immature vessel proliferation following the transplantation of MSC(miR-126) cultures in which the expression of Dll-4 had been knocked down. However, blood vessels with lumen were barely detected, which indicated that Dll-4 plays a key role in tubulogenesis. We conclude that the transplantation of MSCs overexpressing miR-126 can further enhance functional angiogenesis in the ischemic myocardium possibly by the secretion of angiogenic factors and the activation of Dll-4, thus increasing MSC survival. Therefore, MSCs modified with miR-126 may represent a novel and efficient therapeutic approach for ischemic angiogenesis and the improvement of cardiac function.

TLR2 and TLR4 Mediate Differential Responses to Limb Ischemia through MyD88-Dependent and Independent Pathways

IntroductionThe danger signal HMGB1 is released from ischemic myocytes, and mediates angiogenesis in the setting of hindlimb ischemia. HMGB1 is a ligand for innate immune receptors TLR2 and TLR4. While both TLR2 and TLR4 signal through myeloid differentiation factor 88 (MyD88), TLR4 also uniquely signals through TIR-domain-containing adapter-inducing interferon-β (TRIF). We hypothesize that TLR2 and TLR4 mediate ischemic myocyte regeneration and angiogenesis in a manner that is dependent on MyD88 signaling.MethodsMice deficient in TLR2, TLR4, MyD88 and TRIF underwent femoral artery ligation in the right hindlimb. Laser Doppler perfusion imaging was used to assess the initial degree of ischemia and the extent of perfusion recovery. Muscle regeneration, necrosis and fat replacement at 2 weeks post-ligation were assessed histologically and vascular density was quantified by immunostaining. In vitro, endothelial tube formation was evaluated in matrigel in the setting of TLR2 and TLR4 antagonism.ResultsWhile control and TLR4 KO mice demonstrated prominent muscle regeneration, both TLR2 KO and TRIF KO mice exhibited marked necrosis with significant inflammatory cell infiltrate. However, MyD88 KO mice had a minimal response to the ischemic insult with little evidence of injury. This observation could not be explained by differences in perfusion recovery which was similar at two weeks in all the strains of mice. TLR2 KO mice demonstrated abnormal vessel morphology compared to other strains and impaired tube formation in vitro.DiscussionTLR2 and TRIF signaling are necessary for muscle regeneration after ischemia while MyD88 may instead mediate muscle injury. The absence of TLR4 did not affect muscle responses to ischemia. TLR4 may mediate inflammatory responses through MyD88 that are exaggerated in the absence of TLR2. Additionally, the actions of TLR4 through TRIF may promote regenerative responses that are required for recovery from muscle ischemia.

Cell and Gene Therapy Approaches for Cardiac Vascularization

Despite encouraging preclinical results for therapeutic angiogenesis in ischemia, a suitable approach providing sustained, safe and efficacious vascular growth in the heart is still lacking. Vascular Endothelial Growth Factor (VEGF) is the master regulator of angiogenesis, but it also can easily induce aberrant and dysfunctional vascular growth if its expression is not tightly controlled. Control of the released level in the microenvironment around each cell in vivo and its distribution in tissue are critical to induce stable and functional vessels for therapeutic angiogenesis. The present review discusses the limitations and perspectives of VEGF gene therapy and of different cell-based approaches for the implementation of therapeutic angiogenesis in the treatment of cardiac ischemia.

The diverse identity of angiogenic monocytes

The role of bone marrow-derived cells in stimulating angiogenesis, vascular repair or remodelling has been well established, but the nature of the circulating angiogenic cells is still controversial. The existing literature on different cell types that contribute to angiogenesis in multiple pathologies, most notably ischaemic and tumour angiogenesis, is reviewed, with a focus on subtypes of angiogenic mononuclear cells and their local recruitment and activation. A large number of different cells of myeloid origin support angiogenesis without incorporating permanently into the newly formed vessel, which distinguishes these circulating angiogenic cells (CAC) from endothelial progenitor cells (EPC). Although CAC frequently express individual endothelial markers, they all share multiple characteristics of monocytes and only express a limited set of discriminative surface markers in the circulation. When cultured ex vivo, or surrounding the angiogenic vessel in vivo, however, many of them acquire similar additional markers, making their discrimination in situ difficult. Different subsets of monocytes show angiogenic properties, but the distinct microenvironment, in vitro or in vivo, is needed for the development of their pro-angiogenic function.

S100A1 Deficiency Impairs Postischemic Angiogenesis Via Compromised Proangiogenic Endothelial Cell Function and Nitric Oxide Synthase Regulation

Mice lacking the EF-hand Ca2+ sensor S100A1 display endothelial dysfunction because of distorted Ca2+ -activated nitric oxide (NO) generation. To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization. Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue. Ensuing studies in S100A1 knockout (SKO) mice subjected to femoral artery resection unveiled insufficient perfusion recovery and high rates of autoamputation. Defective in vivo angiogenesis prompted cellular studies in SKO ECs and human ECs, with small interfering RNA-mediated S100A1 knockdown demonstrating impaired in vitro and in vivo proangiogenic properties (proliferation, migration, tube formation) and attenuated vascular endothelial growth factor (VEGF)-stimulated and hypoxia-stimulated endothelial NO synthase (eNOS) activity. Mechanistically, S100A1 deficiency compromised eNOS activity in ECs by interrupted stimulatory S100A1/eNOS interaction and protein kinase C hyperactivation that resulted in inhibitory eNOS phosphorylation and enhanced VEGF receptor-2 degradation with attenuated VEGF signaling. Ischemic SKO tissue recapitulated the same molecular abnormalities with insufficient in vivo NO generation. Unresolved ischemia entailed excessive VEGF accumulation in SKO mice with aggravated VEGF receptor-2 degradation and blunted in vivo signaling through the proangiogenic phosphoinositide-3-kinase/Akt/eNOS cascade. The NO supplementation strategies rescued defective angiogenesis and salvaged limbs in SKO mice after femoral artery resection. Our study shows for the first time downregulation of S100A1 expression in patients with critical limb ischemia and identifies S100A1 as critical for EC function in postnatal ischemic angiogenesis. These findings link its pathological plasticity in critical limb ischemia to impaired neovascularization, prompting further studies to probe the microvascular therapeutic potential of S100A1.

P61 Hydrogen sulfide therapy rescues critical limb ischemia in aged diabetic mice through NO/VEGF dependent pathway

Hydrogen sulfide is a gaseous signaling molecule which is produced in vivo by three enzymes such as cystathionine γ-lyase (CSE), cystathionine β- synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MST) from L-cysteine.CSE is the prime enzyme among others for sulfide production in vascular tissues of our body. Hydrogen sulfide is reported to have vasodilatory, anti–inflammatory, anti-oxidant, cytoprotective and pro-angiogenic effects. Sulfide production is decreased in diabetic condition. However, the molecular mechanisms of sulfide-mediated protection during chronic tissue ischemia in diabetic condition are completely unknown. To determine molecular mechanisms involved in hydrogen sulfide mediated cytoprotection during chronic tissue ischemia in diabetic condition. Hind limb ischemia was induced in 10 week old wild type, 42 week old Db/Db diabetic or 10–12 week old CSE knock out (CSEKO, n = 8, each group). PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na 2 S) was administered twice daily by retro-orbital injection. Hind limb perfusion was measured using a laser Doppler perfusion probe. SPY imaging was performed to determine the collateral formation in different time points. Capillary myofiber ratio was determined by NOVA Red staining with CD31 antibody. Mature vessel density and proliferation index were determined by the ratio of smooth muscle actin (SMA) to DAPI and Ki67 to DAPI positive areas respectively. TUNEL assay was measured to investigate the apoptosis. Nitric oxide (NO) was measured by NO analyzer (NOA). CPTIO (1 mg/kg/day) was injected intra-peritoneally to determine the effect of NO in sulfide induced ischemic angiogenesis. VEGF aptamer (50 mg/kg/day) was injected locally to the ischemic muscle to determine the role of VEGF in sulfide induced ischemic angiogenesis. Lastly, VEGF expression was determined by ELISA. Blood perfusion, blush rate by SPY imaging, angiogenic index, proliferation index and mature vessel density were significantly decreased in CSEKO mice compared WT mice indicating the role of sulfide on ischemic angiogeneis and arteriogenesis. Na 2 S dose dependently increases the angiogenesis index, proliferation index, mature vessel density and restores the blood flow in WT mice. Hydrogen sulfide also rescues the impaired blood flow in CSEKO mice by increasing angiogenic index, proliferation index and mature vessel density. Blood tissue perfusion, proliferation index, capillary to myofiber ratio and VEGF expression were all significantly increased and conversely, apoptosis was prevented in ischemic hind limbs of aged diabetic mice treated with Na 2 S compared to PBS control. There was no effect of sulfide on blood glucose, body weight and lipid profiles of diabetic mice.Total NO production was increased in sulfide treated group compared to PBS which was inhibited by CPTIO treatment. Lastly, VEGF164 aptamer blocked sulfide induced augmentation of blood flow and reduced the expression of VEGF164 in WT and diabetic mice hind limb ischemia indicating VEGF164 as the sulfide induced angiogenic stimulator. Sodium sulfide therapy restores tissue perfusion of critical limb ischemia by increasing production of NO, increasing expression of VEGF and preventing apoptosis in diabetic mouse chronic hind limb ischemia which may be beneficial for the diabetic patient with peripheral vascular disease.

Abstract 527: E2F1 Regulates Syndecan-4 Expression Under Hypoxia

The E2F1 transcription factor is known to activate the genes involved in cell cycle progression. However, previous results published by our laboratory showed that the E2F1 plays a crucial role in the regulation of in vivo angiogenesis in mouse hind limb ischemia model. More recently, we demonstrated that E2F1-null (E2F1-/-) mice have a significantly better heart function than wild-type (WT) animals after surgically-induced myocardial infarction (MI). E2F1 -/- mice display reduced endothelial cell (EC) apoptosis, increased EC proliferation, and enhanced angiogenesis in the infarcted heart. To gain mechanistic insights into the role of E2F1 in modulating cellular response to ischemia/hypoxia, we analyzed global gene expression (Illumina microarray) in WT and E2F1 -/- mouse embryonic fibroblasts after treatment with hypoxia (0.5% oxygen) or culture under normoxia for 8 h (n=4). The expression patterns of more than 25,000 genes were analyzed. Among the validated genes syndecan-4, a member of transmembrane proteoglycan family was highly upregulated by hypoxia in E2F1 -/- cells (p&lt;0.01, n=4). Syndecan-4 is a transmembrane heparan sulfate proteoglycan involved in modulation of cell adhesion, migration, growth factor-mediated signaling, and angiogenesis. It is known to possess pro-angiogenic and anti-apoptotic properties, and in humans the levels of syndecan-4 are drastically increased in the border region of damaged cardiac tissue following MI. In mice, loss of syndecan-4 was shown to increase cardiac cell apoptosis, worsen myocardial remodeling, and provoke cardiac rupture after induced ischemic injury. We confirmed our findings by measuring the levels of syndecan-4 expression in the hearts of WT and E2F1 -/- animals after MI. Consistently, we found that the levels of syndecan-4 expression were significantly higher in the E2F1 -/- mice than in WT mice (p&lt;0.05, n=4). Our results suggest that there exist a novel mechanism linking E2F1 and syndecan-4 to the regulation of cardiac ischemic angiogenesis.

Emerging role of PKA/eNOS pathway in therapeutic angiogenesis for ischaemic tissue diseases

Although an abundant amount of research has been devoted to the study of angiogenesis, its precise mechanisms are incompletely understood. Numerous clinical trials focused on therapeutic angiogenesis for the treatment of tissue ischaemia have not been as successful as those of preclinical studies. Thus, additional studies are needed to better understand critical molecular mechanisms regulating ischaemic neovascularization to identify novel therapeutic agents. Nitric oxide (NO) plays a central role in ischaemic neovascularization through the generation of cyclic guanosine monophosphate (cGMP) and the activation of several other signalling responses. Accumulated evidence suggests that endothelial protein kinase A/endothelial NO synthase (PKA/eNOS) signalling may play an important role in ischaemic disorders by promoting neovascularization. This review highlights recent advances in the role of the PKA/eNOS and NO-cGMP-kinase cascade pathway in ischaemic neovascularization. We also discuss molecular relationships of PKA/eNOS with other angiogenic pathways and explore the possibility of activation of the NO/nitrite endocrine system as potential therapeutic targets for ischaemic angiogenesis.

A cellular delivery system fabricated with autologous BMSCs and collagen scaffold enhances angiogenesis and perfusion in ischemic hind limb

Although therapeutic cellular angiogenesis is effective for chronic ischemia, the optimal mode of cellular administration is still under exploration. This study aimed to develop a cellular delivery system to enhance the perfusion and angiogenesis in the ischemic hind limb. Collagen scaffold (CS) was prepared, and for morphology and toxicity analysis, bone marrow-derived mesenchymal stem cells (BMSCs) were isolated, expanded, filtrated, and seeded onto CS to construct BMSCs-CS. The ischemic hind limbs of rabbit models were implanted with autologous BMSCs-CS, CS, and autologous BMSCs; the untreated ischemic or normal animals were considered as the ischemic or normal control groups. Oxygen saturation parameters were regularly measured to determine the perfusion in the extremities. Histological examinations with hematoxylin and eosin immunostaining against von Willebrand factor and smooth muscle (SM) α-actin were performed for capillary and mature vessel evaluation. CS was a multiporous structure without cytotoxicity. At several intervals, the oxygen saturation ratio (OSR) in normal control was the highest. The OSRs in BMSCs-CS and CS were higher than that in BMSCs and ischemic control (p < 0.05); the OSR in BMSCs-CS group was higher than that in CS at 6 and 8 weeks (p < 0.05). The capillaries in BMSCs-CS and CS were higher than that in CS, BMSCs, and the ischemic or normal control (p < 0.05). The mature vessels in BMSCs-CS were higher than that in CS, BMSCs, and the ischemic or normal control (p < 0.05). The autologous cellular delivery system proved to be an effective approach for improving higher ischemic hind limb perfusion and angiogenesis as opposed to cellular therapy alone.

Elevation of hemopexin-like fragment of matrix metalloproteinase-2 tissue levels inhibits ischemic wound healing and angiogenesis

Elevation of hemopexin-like fragment of matrix metalloproteinase-2 tissue levels inhibits ischemic wound healing and angiogenesis

Platelet‐rich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation

The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers.

Elevation of hemopexin-like fragment of matrix metalloproteinase-2 tissue levels inhibits ischemic wound healing and angiogenesis

Matrix metalloproteinase-2 (MMP-2) degrades type IV collagen and enables endothelial cell (EC) migration during angiogenesis and wound healing. Peroxisomal biogenesis factor 2 (PEX2), a by-product of activated MMP-2 autocatalysis, competitively inhibits newly activated MMP-2 from EC surface binding and migration. We hypothesize that PEX2 is elevated during limb ischemia and contributes to poor wound healing, with decreased capillary density. Western blot was used to identify PEX2 in the hind limbs of FVB/NJ mice with surgically induced ischemia. The PEX2 effect on healing was evaluated by calculating the area of exposed muscle after wounding the dorsum of mice and administering daily injections with human recombinant PEX2 (hrPEX2). Wounds were also injected with lentivirus-expressing PEX2 (PEX2-LV), harvested on postoperative day 7 and processed for staining. Epithelial gap was assessed with light microscopy. Capillary density was evaluated after wounding Tie2-green fluorescent protein (GFP)(+) transgenic FVB mice (ECs labeled green) and viral transduction with PEX2-LV. Wounds were harvested on postoperative day (POD) 7, frozen in liquid nitrogen, sectioned, and stained with Hoechst. Vessel density was assessed via fluorescence microscopy as the average number of capillaries/10 high-powered fields. Paired t test was used to assess differences between the groups. PEX2 was elevated 5.5 ± 2.0-fold (P = .005) on POD 2 and 2.9 ± 0.69-fold (P = .004) on POD 4 in gastrocnemius muscles of ischemic hind limbs. The wound surface area, or lack of granulation tissue and exposed muscle, decreased daily in all mice but was greater in the hrPEX2-treated mice by 12% to 16% (P < .004). Wounds in the control group were completely covered with granulation tissue by POD 3. Wounds injected with hrPEX2 were not completely covered by POD 7 but continued to have exposed muscle. Microscopic examination of wounds after PEX2-LV viral transduction demonstrated an average epithelial gap of 1.6 ± 0.3 vs 0.64 ± 0.3 μm in control wounds (P < .04). Wounds from Tie2-GFP mice had an average number of 3.8 ± 1.1 capillaries vs 6.9 ± 1.2 in control wounds (P < .007). Our study links elevated PEX2 to ischemia and poor wound healing. We demonstrate comparative PEX2 elevation in ischemic murine hind limbs. Less granulation tissue is produced and healing is retarded in wounds subjected to hrPEX2 or viral transduction with PEX2-LV. Microscopic examination shows the wounds exhibit fewer capillaries, supporting the hypothesis that PEX2 decreases angiogenesis.

Vascular Protection by Angiotensin Receptor Antagonism Involves Differential VEGF Expression in Both Hemispheres after Experimental Stroke

We identified that the angiotensin receptor antagonist, candesartan, has profound neurovascular protective properties when administered after ischemic stroke and was associated with a proangiogenic state at least partly explained by vascular endothelial growth factor A (VEGFA). However, the spatial distribution of vascular endothelial growth factor (VEGF) isoforms and their receptors remained unknown. Protein analysis identified a significant increase in vascular endothelial grow factor B (VEGFB) in the cerebrospinal fluid (CSF) and the ischemic hemispheres (with increased VEGF receptor 1 activation) of treated animals (p<0.05) which was co-occurring with an increase in protein kinase B (Akt) phosphorylation (p<0.05). An increase in VEGFA protein in the contralesional hemisphere corresponded to a significant increase in vascular density at seven days (p<0.01) after stroke onset. Vascular restoration by candesartan after stroke maybe related to differential regional upregulation of VEGFB and VEGFA, promoting a “prosurvival state” in the ischemic hemisphere and angiogenesis in the contralesional side, respectively. These vascular changes in both hemispheres after effective treatment are likely to contribute to enhanced recovery after stroke.

A new strategy of promoting vascularization of skin substitutes by capturing endothelial progenitor cells automatically

How to promote vascularization of a skin substitute is the key to successful skin transplantation. Current methods are mainly through releasing angiogenesis-related factors (ARF) or seeding angiogenesis-related cells (ARC), but the efficacy of these methods is not satisfactory, because angiogenesis needs participation of multiple factors, extracellular matrix and related cells. The latest research has demonstrated that endothelial progenitor cells (EPCs) originating from bone marrow and existing in peripheral blood are the key element participating in revascularization of adult tissues. They directly participate in both stem cell vasculogenesis of ischemic tissues and local angiogenesis. We therefore hypothesize whether it is possible to construct a new skin substitute and use it to mobilize EPCs in bone marrow to peripheral circulation and capture EPCs automatically as a simple and effective method of promoting vascularization of the skin substitute for the sake of improving its post-transplant survival.

Impaired Expression of Uncoupling Protein 2 Causes Defective Postischemic Angiogenesis in Mice Deficient in AMP-Activated Protein Kinase α Subunits

The aim of the present study was to determine whether mitochondrial uncoupling protein (UCP) 2 is required for AMPK-dependent angiogenesis in ischemia in vivo. Angiogenesis was assayed by monitoring endothelial tube formation (a surrogate for angiogenesis) in human umbilical vein endothelial cells (ECs), isolated mouse aortic endothelial cells (MAECs), and pulmonary microvascular endothelial cells or in ischemic thigh adductor muscles from wild-type (WT) mice or mice deficient in either AMPKα1 or AMPKα2. AMPK inhibition with pharmacological inhibitor (compound C) or genetic means (transfection of AMPKα-specific small interfering RNA) significantly lowered the tube formation in human umbilical vein ECs. Consistently, compared with WT mice, tube formation in MAECs isolated from either AMPKα1(-/-) or AMPKα2(-/-) mice, which exhibited oxidative stress and reduced expression of UCP2, was significantly impaired. In addition, adenoviral overexpression of UCP2, but not adenoviruses encoding green fluorescent protein, normalized tube formation in MAECs from either AMPKα1(-/-) or AMPKα2(-/-) mice. Similarly, supplementation with sodium nitroprusside, a nitric oxide (NO) donor, restored tube formation. Furthermore, ischemia significantly increased angiogenesis, serine 1177 phosphorylation of endothelial NO synthase, and UCP2 in ischemic thigh adductor muscles from WT mice but not in those from either AMPKα1(-/-) or AMPKα2(-/-) mice. We conclude that AMPK-dependent UCP2 expression in ECs promotes angiogenesis in vivo.