Angiogenesis In Skeletal Muscle Research Articles

Hydrogen Sulfide Improves Hyperhomocysteinemia‐Mediated Impairment of Angiogenesis in Skeletal Muscle

The growth of new blood vessels (angiogenesis) is a fundamental process of human health to satisfy energy requirements, tissue growth, and wound healing. Although hyperhomocysteinemia (HHcy) (~5% of general population) is associated with myopathy, the precise molecular mechanism(s) remain unknown. In this study, we examined the potential effects of HHcy on impairment of angiogenesis in skeletal muscle by antagonizing the angiogenic signals via PI3K/AKT pathway. We used skeletal muscle from WT (C57BL/6J) and HHcy CBS (+/−) mouse model (8 to 10 weeks) and hind limb ischemia were carried out via femoral artery ligation to study the process of angiogenesis. Mice were treated with the H2S donor: NaHS (10 mg/kg) intraperitoneally. After 7, 14 and 21 days of hindlimb ischemia surgery, barium sulfate angiography, immune staining for CD31 and laser Doppler blood flow measurement were carried out in skeletal muscle of both ischemic and the contralateral leg (sham control). Whereas, the expression of all angiogenic factors was analyzed by angiogenic protein array and subsequently confirmed by western blots analysis. We found that the vessel density was significantly less in post-surgery CBS mice compared to WT mice and these effects of HHcy were mitigated via NaHS treatment in a time-dependent manner. The levels of angiogenic factors like hypoxia inducing factor (HIF1-α), growth factors (VEGF), the vasodilator (NO) and structural protein (dystrophin) was significantly attenuated in skeletal muscle of CBS mice in comparison to WT mice, which was improved by NaHS treatment. We also noticed the Akt-phosphorylation was decreased in CBS mice compared to WT mice. These results suggest that HHcy can inhibit angiogenesis via antagonizing the angiogenic signals by interfering PI3K/AKT pathway and NaHS treatment can mitigate these effects of HHcy. We are currently studying whether the outlined processes resulted in myopathy via poor angiogenic development in skeletal muscle during HHcy. Support or Funding Information This work was supported, in part, by a grant from the National Institute of Health (Heart, Lung, and Blood Institute; No. HL-74815). Proposed model This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Topical Icing on Inflammation, Angiogenesis, Revascularization, and Myofiber Regeneration in Skeletal Muscle Following Contusion Injury.

Contusion injuries in skeletal muscle commonly occur in contact sport and vehicular and industrial workplace accidents. Icing has traditionally been used to treat such injuries under the premise that it alleviates pain, reduces tissue metabolism, and modifies vascular responses to decrease swelling. Previous research has examined the effects of icing on inflammation and microcirculatory dynamics following muscle injury. However, whether icing influences angiogenesis, collateral vessel growth, or myofiber regeneration remains unknown. We compared the effects of icing vs. a sham treatment on the presence of neutrophils and macrophages; expression of CD34, von Willebrands factor (vWF), vascular endothelial growth factor (VEGF), and nestin; vessel volume; capillary density; and myofiber regeneration in skeletal after muscle contusion injury in rats. Muscle tissue was collected 1, 3, 7, and 28 d after injury. Compared with uninjured rats, muscles in rats that sustained the contusion injury exhibited major necrosis, inflammation, and increased expression of CD34, vWF, VEGF, and nestin. Compared with the sham treatment, icing attenuated and/or delayed neutrophil and macrophage infiltration; the expression of vWF, VEGF, and nestin; and the change in vessel volume within muscle in the first 7 d after injury (P < 0.05). By contrast, icing did not influence capillary density in muscle 28 d after injury (P = 0.59). The percentage of immature myofibers relative to the total number of fibers was greater in the icing group than in the sham group 28 d after injury (P = 0.026), but myofiber cross-sectional area did not differ between groups after 7 d (P = 0.35) and 28 d (P = 0.30). In conclusion, although icing disrupted inflammation and some aspects of angiogenesis/revascularization, these effects did not result in substantial differences in capillary density or muscle growth.

Blunted transcriptional response to skeletal muscle ischemia in rats with chronic kidney disease: potential role for impaired ischemia-induced angiogenesis.

Chronic kidney disease (CKD) is associated with increased cardiovascular morbidity and mortality. Previous studies indicated an impairment of ischemia-induced angiogenesis in skeletal muscle of rats with CKD. We performed a systematic comparison of early gene expression in response to ischemia in rats with or without CKD to identify potential molecular mechanisms underlying impaired angiogenesis in CKD. CKD was induced in male rats by 5/6 nephrectomy (SNX); control rats were sham operated (sham). Eight weeks later, ischemia of the right limb was induced by ligation and resection of the femoral artery. Rats were killed 24 h after the onset of ischemia, and RNA was extracted from the musculus soleus of the ischemic and the nonischemic hindlimb. To identify differentially expressed transcripts, we analyzed RNA with Affymetrix GeneChip Rat Genome 230 2.0 Arrays. RT-PCR analysis of selected genes was performed to validate observed changes. Hindlimb ischemia upregulated 239 genes in CKD and 299 genes in control rats (66% overlap), whereas only a few genes were downregulated (14 in CKD and 34 in controls) compared with the nonischemic limb of the same animals. Comparison between the ischemic limbs of CKD and controls revealed downregulation of 65 genes in CKD; 37 of these genes were also among the ischemia-induced genes in controls. Analysis of functional groups (other than angiogenesis) pointed to genes involved in leukocyte recruitment and fatty acid metabolism. Transcript expression profiling points to a relatively small number of differentially expressed genes that may underlie the impaired postischemic angiogenesis in CKD.

Four-Dimensional Microvascular Analysis Reveals That Regenerative Angiogenesis in Ischemic Muscle Produces a Flawed Microcirculation.

Angiogenesis occurs after ischemic injury to skeletal muscle, and enhancing this response has been a therapeutic goal. However, to appropriately deliver oxygen, a precisely organized and exquisitely responsive microcirculation must form. Whether these network attributes exist in a regenerated microcirculation is unknown, and methodologies for answering this have been lacking. To develop 4-dimensional methodologies for elucidating microarchitecture and function of the reconstructed microcirculation in skeletal muscle. We established a model of complete microcirculatory regeneration after ischemia-induced obliteration in the mouse extensor digitorum longus muscle. Dynamic imaging of red blood cells revealed the regeneration of an extensive network of flowing neo-microvessels, which after 14 days structurally resembled that of uninjured muscle. However, the skeletal muscle remained hypoxic. Red blood cell transit analysis revealed slow and stalled flow in the regenerated capillaries and extensive arteriolar-venular shunting. Furthermore, spatial heterogeneity in capillary red cell transit was highly constrained, and red blood cell oxygen saturation was low and inappropriately variable. These abnormalities persisted to 120 days after injury. To determine whether the regenerated microcirculation could regulate flow, the muscle was subjected to local hypoxia using an oxygen-permeable membrane. Hypoxia promptly increased red cell velocity and flux in control capillaries, but in neocapillaries, the response was blunted. Three-dimensional confocal imaging revealed that neoarterioles were aberrantly covered by smooth muscle cells, with increased interprocess spacing and haphazard actin microfilament bundles. Despite robust neovascularization, the microcirculation formed by regenerative angiogenesis in skeletal muscle is profoundly flawed in both structure and function, with no evidence for normalizing over time. This network-level dysfunction must be recognized and overcome to advance regenerative approaches for ischemic disease.

Obesity Downregulates MicroRNA-126 Inducing Capillary Rarefaction in Skeletal Muscle: Effects of Aerobic Exercise Training.

Background. We investigated the effects of exercise training (ET) on miR-126 levels and skeletal muscle angiogenesis in obese Zucker rats. Results. Zucker rats were randomly assigned to sedentary and swimming-trained groups: lean sedentary (LS) and trained (LTR); obese sedentary (OB) and trained (OBTR). The OB group displayed capillary rarefaction compared with the LS group. In contrast, ET increased the capillary/fiber ratio by 38% in the LTR group and normalized capillary rarefaction in the OBTR group. VEGF, PI3K, and eNOS levels were reduced in the skeletal muscle of the OB group. ET normalized VEGF, PI3K, and eNOS levels in OBTR, contributing to vascular network homeostasis. PI3KR2 inhibits PI3K, a key mediator of the VEGF signaling pathway. Obesity decreased miR-126 and increased PI3KR2 levels compared with the LS group. However, ET normalized miR-126 levels in the OBTR group versus the LS group and decreased expression of PI3KR2. Conclusion. Our findings show that obesity leads to skeletal muscle capillary rarefaction, which is regulated by decreased miR-126 levels and increased PI3KR2. Inversely, ET normalizes miR-126 levels and VEGF signaling and should be considered an important therapeutic strategy for vascular disorders.

Prazosin Can Prevent Glucocorticoid Mediated Capillary Rarefaction

Glucocorticoids (GC) elicit skeletal muscle capillary rarefaction, which can subsequently impair blood distribution and muscle function; however, the mechanisms have not been established. We hypothesized that CORT would inhibit endothelial cell survival signals but that treatment with the alpha-1 adrenergic receptor inhibitor prazosin, which leads to angiogenesis in skeletal muscle of healthy rats, would reverse these effects and induce angiogenesis within the skeletal muscle of corticosterone (CORT)-treated rats. Male Sprague Dawley rats were implanted subcutaneously with CORT pellets (400 mg/rat), with or without concurrent prazosin treatment (50mg/L in drinking water), for 1 or 2 weeks. Skeletal muscle capillary rarefaction, as indicated by a significant reduction in capillary-to-fiber ratio (C:F), occurred after 2 weeks of CORT treatment. Concurrent prazosin administration prevented this capillary rarefaction in CORT-treated animals but did not induce angiogenesis or arteriogenesis as was observed with prazosin treatment in control rats. CORT treatment reduced the mRNA level of Angiopoietin-1 (Ang-1), which was partially offset in the muscles of rats that received 2 weeks of co-treatment with prazosin. In 2W CORT animals, prazosin treatment elicited a significant increase in vascular endothelial growth factor-A (VEGF-A) mRNA and protein. Conversely prazosin did not rescue CORT-induced reductions in transforming growth factor beta-1 (TGFβ1 and matrix metalloproteinase-2 (MMP-2) mRNA. To determine if CORT impaired shear stress dependent signaling, cultured rat skeletal muscle endothelial cells were pre-treated with CORT (600nM) for 48 hours, then exposed to 15 dynes/cm2 shear stress or maintained with no flow. CORT blunted the shear stress-induced increase in pSer473 Akt, while pThr308 Akt, ERK1/2 and p38 phosphorylation and nitric oxide (NO) production were unaffected. This study demonstrates that GC-mediated capillary rarefaction is associated with a reduction in Ang-1 mRNA within the skeletal muscle microenvironment and that concurrent prazosin treatment effectively increases VEGF-A levels and prevents capillary loss.

The Vascular Endothelial Growth Factor-A phosphorylates Murine Double Minute-2 on its Serine 166 via the Extracellular Signal-Regulated Kinase 1/2 and p90 Ribosomal S6 Kinase in primary human endothelial cells

Murine Double Minute-2 (Mdm2) has been identified as an essential regulator of skeletal muscle angiogenesis and the pro-angiogenic activity of endothelial cells. We have recently demonstrated that the pro-angiogenic Vascular Endothelial Growth Factor-A (VEGF-A) is a potent upstream regulator of Mdm2 phosphorylation on its Serine 166 (p-Ser166-Mdm2), a protein modification leading to an increase in endothelial cell migration. Here, we investigated the kinase signaling pathways that could be responsible for mediating VEGF-A-dependent Mdm2 phosphorylation. Incubation of primary human dermal microvascular endothelial cells with recombinant VEGF-A for 15 min led to increased phosphorylation levels of VEGF-receptor-2, Mdm2, Akt, Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), and p90 Ribosomal S6 Kinase (p90RSK) proteins. In addition to being linked to VEGF-A signaling, Akt, ERK1/2 and p90RSK have been shown to potentially lead to Mdm2 phosphorylation. We therefore next analyzed which of these kinases could be responsible for VEGF-A-dependent Mdm2 phosphorylation on Serine 166 by using kinase-specific pharmacological inhibitors. Inhibition of ERK1/2 phosphorylation by UO126 entirely abrogated the response of p-Ser166-Mdm2 to VEGF-A treatment, while Akt phosphorylation inhibition by wortmannin led to further elevations in p-Ser166-Mdm2. p90RSK has been identified as a potential candidate downstream of ERK1/2 that could induce Mdm2 Ser166 phosphorylation. Two independent p90RSK inhibitors, FMK and BI-D1870, each led to an entire loss of p-Ser166-Mdm2 responsiveness to VEGF-A. Taken together, our results demonstrate that VEGF-A driven Mdm2 phosphorylation on Ser166 is dependent on the ERK1/2/p90RSK signaling pathway in primary human endothelial cells, furthering our understanding of the complex relationship between Mdm2 and VEGF-A in a physiological context.

Morinda citrifolia leaf enhanced performance by improving angiogenesis, mitochondrial biogenesis, antioxidant, anti-inflammatory & stress responses

Morinda citrifolia fruit, (noni), enhanced performances in athletes and post-menopausal women in clinical studies. This report shows the edible noni leaves water extract enhances performance in a weight-loaded swimming animal model better than the fruit or standardized green tea extract. The 4weeks study showed the extract (containing scopoletin and epicatechin) progressively prolonged the time to exhaustion by threefold longer than the control, fruit or tea extract. The extract improved (i) the mammalian antioxidant responses (MDA, GSH and SOD2 levels), (ii) tissue nutrient (glucose) and metabolite (lactate) management, (iii) stress hormone (cortisol) regulation; (iv) neurotransmitter (dopamine, noradrenaline, serotonin) expressions, transporter or receptor levels, (v) anti-inflammatory (IL4 & IL10) responses; (v) skeletal muscle angiogenesis (VEGFA) and (v) energy and mitochondrial biogenesis (via PGC, UCP3, NRF2, AMPK, MAPK1, and CAMK4). The ergogenic extract helped delay fatigue by enhancing energy production, regulation and efficiency, which suggests benefits for physical activities and disease recovery.

Differential Expansion of the Capillary Bed through Physiological Angiogenesis in Skeletal Muscle

A range of well‐established in vivo models of angiogenesis currently exists, involving surgical and pharmacological intervention. These approaches generally improve global measures of microvascular supply, but are rarely interrogated for their capacity to improve tissue oxygen tension. The aim of this research was to characterise a variety of muscle recruitment patterns known to induce angiogenesis, determine their effect on capillary spatial heterogeneity, and subsequently local tissue oxygen delivery.Male Wistar rats underwent seven days of angiogenic remodelling through indirect electrical stimulation of skeletal muscle (at 4, 10 or 40Hz), muscular overload (through synergist ablation) or pharmacological supplementation (Prazosin, alpha‐adrenergic blocker). Muscle samples were snap frozen and capillary content identified through Griffonia simplicifolia lectin staining, with morphometric analysis of muscle composition used as the basis for oxygen consumption modelling.We demonstrated that seven days of supramaximal indirect electrical stimulation induced significant angiogenesis (capillary to fibre ratio (C:F) increased by 59, 36 and 45% for 4, 10 and 40Hz, respectively). A similar response for muscular overload and prazosin supplementation were seen with a 42 and 27% increase, respectively. This improvement in capillarity was accompanied by a reduction in mean capillary domain size (tissue supply area), but with varying degrees of spatial heterogeneity.Skeletal muscle overload and 40Hz electrical stimulation showed a substantially more homogeneous capillary distribution, and subsequently improved tissue oxygenation at high levels of simulated exercise. Conversely, the improved capillarity associated with prazosin and 4Hz electrical stimulation showed no extensive change in capillary distribution. We therefore conclude that mechanical stimulation of angiogenesis may be accentuated by a metabolic component.

Angiotensin‐(1–7) Requires both Mas1 and AT1 Receptors to Restore Angiogenesis

The Renin‐Angiotensin System plays an important role in many physiological processes such as sodium homeostatis, regulating vascular tone and angiogenesis. The octapeptide Angiotensin II (AngII) acting through the AT1 receptor has long been known to promote vasoconstriction, fibrosis, cell proliferation and skeletal muscle angiogenesis in response to exercise or electrical stimulation. A second vasoactive peptide of the Renin‐Angiotensin System, Angiotensin‐1–7 (Ang‐(1–7)), acts through the Mas1 receptor and is generally thought to have many physiological actions that are antagonistic to the actions of the AngII‐AT1 axis, such as vasodilation, anti‐fibrotic and anti‐proliferative effects. We hypothesized Ang‐(1–7) acting through the Mas1 receptor could restore skeletal muscle angiogenesis in response to electrical stimulation. Little is known about the signaling events initiated by Mas1 receptor activation by Ang‐(1–7) relative to AngII‐AT1 activity. Here we show in Sprague Dawley rats with Renin suppressed through high dietary salt intake, which suppresses angiogenesis, that infused Ang‐(1–7) acting through the Mas1 receptor restores angiogenesis in vivo in skeletal muscle in response to electrical stimulation and in vitro in a tube formation assay using rat microvascular endothelial cells, in both cases independent of AngII. Using advanced proteomic methodology to characterize the components of Mas1 receptor signaling we identified signaling pathways that may play a role in the observed angiogenic response, such as nascent protein synthesis, exocytosis, cellular reorganization and regulators such as NO, NF‐kappaB and ERK1/2 signaling. Inhibition of ERK1/2 signaling inhibited tube formation by endothelial cells stimulated by AngII or Ang‐(1–7). We further assessed angiogenesis in response to electrical stimulation promoted by Ang‐(1–7) in SS‐Mas1em1Mcwi (Mas1 mutant knockout) and SS‐Agtr1aem5Mcwi (AT1 mutant knock out) strains, and compared to wild‐type SS/Mcwi. We found reduced angiogenesis to electrical stimulation after Ang‐(1–7) or orally active Ang‐(1–7) analog AVE0991 treatment in both the Mas1 mutant and the AT1 mutant knock outs relative to the SS/Mcwi. The RAS is a complex physiological system that can affect a wide range of processes in the cardiovascular system. Even though the AT1 and Mas1 receptor axes largely have antagonistic functions, we have shown that capillary angiogenesis can both be restored by AngII‐AT1 or Ang‐(1–7)‐Mas1 activity, and the restoration depends on the presence of functioning Mas1 and AT1 receptors.Support or Funding InformationNIH Heart, Lung and Blood Institute 082798

(-)-Epicatechin Attenuates Degradation of Mouse Oxidative Muscle Following Hindlimb Suspension.

The purpose of this study was to conduct a 14-day hindlimb suspension (HS) with and without (-)-epicatechin supplementation to determine whether (-)-epicatechin treatment can attenuate the loss in muscle degradation, angiogenesis, and mitochondrial signaling in oxidative skeletal muscle. Adult mice were randomized into 3 groups: (a) control (C); (b) HS with vehicle (HS-V); and (c) HS with (-)-epicatechin (HS-(-)-Epi). Animals in the HS-(-)-Epi group received (-)-epicatechin (1.0 mg · kg(-1) of body mass) twice daily through oral gavage. For markers related to muscle degradation, the HS-V group had significantly higher protein expression compared with the control and HS-(-)-Epi groups. Moreover, protein expression for myosin heavy chain type I was significantly reduced by approximately 45% in the HS-V group compared with the control and HS-(-)-Epi groups. In addition, capillarity contact and capillary-to-fiber ratio were significantly higher in the HS-(-)-Epi group compared with the HS-V group. Furthermore, protein expression for thrombospondin-1 was significantly higher in HS-V group compared with the control and HS-(-)-Epi groups. Hindlimb suspension also significantly reduced protein expression for mitochondrial signaling compared with the control and HS-(-)-Epi groups. These findings suggest that (-)-epicatechin supplementation attenuates degradation in oxidative muscles after HS.

PGC-1α-mediated changes in phospholipid profiles of exercise-trained skeletal muscle

Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α. We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle.

Advances and challenges in skeletal muscle angiogenesis.

The role of capillaries is to serve as the interface for delivery of oxygen and removal of metabolites to/from tissues. During the past decade there has been a proliferation of studies that have advanced our understanding of angiogenesis, demonstrating that tissue capillary supply is under strict control during health but poorly controlled in disease, resulting in either excessive capillary growth (pathological angiogenesis) or losses in capillarity (rarefaction). Given that skeletal muscle comprises nearly 40% of body mass in humans, skeletal muscle capillary density has a significant impact on metabolism, endocrine function, and locomotion and is tightly regulated at many different levels. Skeletal muscle is also high adaptable and thus one of the few organ systems that can be experimentally manipulated (e.g., by exercise) to study physiological regulation of angiogenesis. This review will focus on the methodological concerns that have arisen in determining skeletal muscle capillarity and highlight the concepts that are reshaping our understanding of the angio-adaptation process. We also summarize selected new findings (physical influences, molecular changes, and ultrastructural rearrangement of capillaries) that identify areas of future research with the greatest potential to expand our understanding of how angiogenesis is normally regulated, and that may also help to better understand conditions of uncontrolled (pathological) angiogenesis.

Phosphorylation of murine double minute-2 on Ser166 is downstream of VEGF-A in exercised skeletal muscle and regulates primary endothelial cell migration and FoxO gene expression.

We demonstrated in a previous study that murine double minute (Mdm)-2 is essential for exercise-induced skeletal muscle angiogenesis. In the current study, we investigated the mechanisms that regulate Mdm2 activity in response to acute exercise and identified VEGF-A as a key stimulator of Mdm2 phosphorylation on Ser(166) (p-Ser166-Mdm2). VEGF-A and p-Ser166-Mdm2 protein levels were measured in human and rodent muscle biopsy specimens after 1 bout of exercise. VEGF-A-dependent Mdm2 phosphorylation was demonstrated in vivo in mice harboring myofiber-specific deletion of VEGF-A (mVEGF(-/-)) and in vitro in primary human and rodent endothelial cells (ECs). Exercise increased VEGF-A and p-Ser166-Mdm2 protein levels respectively by 157 and 68% in human muscle vs. pre-exercise levels. Similar results were observed in exercised rodent muscles compared to sedentary controls; however, exercise-induced Mdm2 phosphorylation was significantly attenuated in mVEGF(-/-) mice. Recombinant VEGF-A elevated p-Ser166-Mdm2 by 50-125% and stimulated migration by 33% in ECs when compared to untreated cells, whereas the Mdm2 antagonist Nutlin-3a abrogated VEGF-driven EC migration. Finally, overexpression of a Ser166-Mdm2 phosphorylation mimetic increased EC migration, increased Mdm2 to FoxO1 binding (+55%), and decreased FoxO1-dependent gene expression compared with ECs overexpressing WT-Mdm2. Our results suggest that VEGF-mediated Mdm2 phosphorylation on Ser(166) is a novel proangiogenic pathway within the skeletal muscle.

Abstract 16016: Role For Macitentan in Angiogenesis of Right Ventricle, Peripheral Muscle and Lungs in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a vascular disease characterized by remodeling of distal pulmonary arteries (PA) leading to increased mean PA pressure, subsequent right ventricular (RV) failure and ultimately death of patients. Impaired angiogenesis is now recognized as a major factor in PAH etiology. It contributes not only to the decrease in lung perfusion but also to a rarefaction of the capillary network within the skeletal muscle and RV leading to exercise intolerance and RV failure. Several studies have suggested that endothelin-1 (ET-1) contributes to decrease angiogenesis in PAH. In light of these observations, we hypothesized that macitentan, an ET-1 receptors blocker, improves PAH by promoting angiogenesis in lungs, RV and skeletal muscles. Methods/Results: In the Sugen/hypoxia rat model of PAH, we demonstrated by right catheterization in closed chest rats that compared to vehicle treated animals, daily treatment during two weeks with macitentan (30 mg/kg) decreases significantly mean PA pressure (n=8-20; p&lt;0.01) and total pulmonary resistance (n=8-20; p&lt;0.05) and improves cardiac output, stroke volume (n=7-17; p&lt;0.05) and RV hypertrophy measured by echo (n=6-10; p&lt;0.05). These effects were associated with an increase of 2.9 fold in lung blood perfusion (n=6), 4.1 fold in quadriceps perfusion (n=5-7) and 1.5 fold in right ventricle perfusion (n=5-6) measured in vivo by micro CT angiogram (p&lt;0.05). Both in vivo lectin perfusion (n=4-10; p&lt;0.05) and immunologic CD31 staining (n=4-5; p&lt;0.05) confirmed these findings. In the lungs, the hemodynamic and perfusion improvements were associated with decreased vascular remodeling (H&amp;E) (n=6-17; p&lt;0.01). In vitro, using primary cultured human endothelial cells isolated from distal PA (PAEC) and RV (RVEC), we showed that macitentan decreases PAH-PAEC proliferation (Ki67 assay) and resistance to apoptosis (TUNEL assay), while increasing angiogenesis capacity of healthy PAEC and RVEC (matrigel assay). Conclusion: Our data provide structural and functional evidence that ET-1 receptor inhibition with macitentan may improve PAH by promoting angiogenesis.

Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165.

IntroductionCell therapy using adipose-derived stromal cells (ADSC) is an intensively developing approach to promote angiogenesis and regeneration. Administration technique is crucial and among others minimal constructs - cell sheets (CS) have certain advantages. Delivery of CS allows transplantation of cells along with matrix proteins to facilitate engraftment. Cells’ therapeutic potential can be also increased by expression of proangiogenic factors by viral transduction. In this work we report on therapeutic efficacy of CS from mouse ADSC transduced to express human vascular endothelial growth factor 165 a/a isoform (VEGF165), which showed potency to restore perfusion and protect tissue in a model of limb ischemia.MethodsMouse ADSC (mADSC) isolated from C57 male mice were expanded for CS formation (106cells per CS). Constructs were transduced to express human VEGF165 by baculoviral (BV) system. CS were transplanted subcutaneously to mice with surgically induced limb ischemia and followed by laser Doppler perfusion measurements. At endpoint animals were sacrificed and skeletal muscle was evaluated for necrosis and vessel density; CS with underlying muscle was stained for apoptosis, proliferation, monocytes and blood vessels.ResultsUsing BV system and sodium butyrate treatment we expressed human VEGF165 in mADSC (production of VEGF165 reached ≈ 25-27 ng/ml/105 cells) and optimized conditions to ensure cells’ viability after transduction. Implantation of mock-transduced CS resulted in significant improvement of limb perfusion, increased capillary density and necrosis reduction at 2 weeks post-surgery compared to untreated animals. Additional improvement of blood flow and angiogenesis was observed after transplantation of VEGF165-expressing CS indicating enhanced therapeutic potential of genetically modified constructs. Moreover, we found delivery of mADSC as CS to be superior to equivalent dose of suspended cells in terms of perfusion and angiogenesis. Histology analysis of extracted CS detected limited proliferation and approximately 10 % prevalence of apoptosis in transplanted mADSC. Significant vascularization of CS and infiltration by monocytes were found in both – BV-transduced and control CS indicating graft and host interaction after transplantation.ConclusionsDelivery of ADSC by subcutaneous transplantation of CS is effective for stimulation of angiogenesis and tissue protection in limb ischemia with a potential for efficacy improvement by BV transduction to express VEGF165.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0199-6) contains supplementary material, which is available to authorized users.

Angiogenesis-related ultrastructural changes to capillaries in human skeletal muscle in response to endurance exercise.

The ultrastructure of capillaries in skeletal muscle was morphometrically assessed in vastus lateralis muscle (VL) biopsies taken before and after exercise from 22 participants of two training studies. In study 1 (8 wk of ergometer training), light microscopy revealed capillary-fiber (C/F) ratio (+27%) and capillary density (+16%) to be higher (P ≤ 0.05) in postexercise biopsies than in preexercise biopsies from all 10 participants. In study 2 (6 mo of moderate running), C/F ratio and capillary density were increased (+23% and +20%; respectively, P ≤ 0.05) in VL biopsies from 6 angiogenesis responders (AR) after training, whereas 6 nonangiogenesis responders (NR) showed nonsignificant changes in these structural indicators (-4%/-4%, respectively). Forty capillary profiles per participant were evaluated by point and intersection counting on cross sections after transmission electron microscopy. In study 1, volume density (Vv) and mean arithmetic thickness (T) of endothelial cells (ECs; +19%/+17%, respectively) and pericytes (PCs; +20%/+21%, respectively) were higher (P ≤ 0.05), whereas Vv and T of the pericapillary basement membrane (BM) were -23%/-22% lower (P ≤ 0.05), respectively, in posttraining biopsies. In study 2, exercise-related differences between AR and NR-groups were found for Vv and T of PCs (AR, +26%/+22%, respectively, both P ≤ 0.05; NR, +1%/-3%, respectively, both P > 0.05) and BM (AR, -14%/-13%, respectively, both P ≤ 0.05; NR, -9%/-11%, respectively, P = 0.07/0.10). Vv and T of ECs were higher (AR, +16%/+18%, respectively; NR, +6%/+6%, respectively; all P ≤ 0.05) in both groups. The PC coverage was higher (+13%, P ≤ 0.05) in VL biopsies of individuals in the AR group but nonsignificantly altered (+3%, P > 0.05) in those of the NR group after training. Our study suggests that intensified PC mobilization and BM thinning are related to exercise-induced angiogenesis in human skeletal muscle, whereas training per se induces EC-thickening.

Transplantation of cell sheets from adipose-derived mesenchymal stromal cells effectively induces angiogenesis in ischemic skeletal muscle

Transplantation of cell sheets from adipose-derived mesenchymal stromal cells effectively induces angiogenesis in ischemic skeletal muscle

Subthreshold High-Frequency Electrical Field Stimulation Induces VEGF Expression in Cardiomyocytes.

Subthreshold electrical stimulation (SES) has been shown to induce an improvement of angiogenesis in ischemic and nonischemic skeletal muscles, mediated by increased VEGF expression. VEGF plays a key role in physiological and pathological angiogenesis. Cardiomyocytes possess the ability to synthesize and secrete VEGF. Thus, we thought to investigate the effect of SES on VEGF regulation in cultured neonatal rat ventricular myocytes (NRVMs), in the aim to reveal new techniques for therapeutic angiogenesis in ischemic heart disease. Cell cultures of NRVMs were electrically stimulated with field strengths below the myocyte depolarization threshold (0.5 V/cm with 1 ms bipolar impulse duration). Frequencies ranging from 5 Hz up to 25, 50, and 99 Hz were applied over a period of 48 h. The expression of VEGF and its receptor KDR was determined with Western blot and ELISA. To reveal the biological activity of the secreted VEGF amount, cultured human coronary artery endothelial cells (HCAECs) were treated with the cell culture supernatant of NRVMs exposed to SES. A dominant effect of SES was observed at 25 Hz. Within this particular frequency the VEGF protein amount in the cytoplasm as well as in the cell culture supernatant increased significantly. In parallel, the protein expression of the KDR receptor decreased in a significant manner. Moreover, cell culture supernatant of NRVMs exposed to SES augmented the growth of HCAECs. Cardiomyocytes respond to SES with an increase in biologically active VEGF expression that promotes cell proliferation of HCAECs. This mechanism may provide new approaches to develop therapeutic angiogenesis in the ischemic heart.

What turns off the angiogenic switch in skeletal muscle?

What turns off the angiogenic switch in skeletal muscle?