Anti-angiogenic Thrombospondin-1 Research Articles

Pericardial Adipose Tissue Thrombospondin-1 Associates With Antiangiogenesis in Ischemic Heart Disease

Accumulation of ectopic pericardial adipose tissue has been associated with cardiovascular complications which, in part, may relate to adipose-derived factors that regulate vascular responses and angiogenesis. We sought to characterize adipose tissue microvascular angiogenic capacity in individuals undergoing elective cardiac surgeries including aortic, valvular, and coronary artery bypass grafting. Pericardial adipose tissue was collected intraoperatively and examined for angiogenic capacity. Capillary sprouting was significantly blunted (2-fold, p<0.001) in subjects with CAD (age 60±9 years, BMI 32±4 kg/m2, LDL-C 95±46 mg/dl, n=29) compared to age-, BMI-, and LDL-C matched individuals without angiographic obstructive CAD (age 59±10 years, BMI 35±9 kg/m2, LDL-C 101±40 mg/dl, n=12). For potential mechanistic insight, we performed mRNA expression analyses using quantitative RT- PCR, and observed no significant differences in pericardial fat gene expression of pro-angiogenic mediators vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and angiopoietin-1 (angpt1), or anti-angiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and endostatin. In contrast, mRNA expression of anti-angiogenic thrombospondin-1 (TSP-1) was significantly upregulated (2-fold, p=0.008) in CAD compared to non-CAD subjects, which was confirmed by protein western-immunoblot analysis. TSP-1 gene knockdown using shRNA lentiviral delivery significantly improved angiogenic deficiency in CAD (p<0.05). In conclusion, pericardial fat in subjects with CAD may be associated with an anti-angiogenic profile linked to functional defects in vascularization capacity. Local paracrine actions of TSP-1 in adipose depots surrounding the heart may play a role in mechanisms of ischemic heart disease.

Abstract 14820: Pericardial Adipose Tissue Thrombospondin-1 Associates With Anti-Angiogenesis in Ischemic Heart Disease

Defective angiogenesis contributes to mechanisms of ischemic heart disease. Accumulation of ectopic pericardial adipose tissue has been associated with cardiovascular complications which, in part, may relate to adipose tissue-derived factors. We sought to characterize adipose tissue microvascular angiogenic capacity in individuals undergoing elective cardiac surgeries including aortic, valvular, and coronary artery bypass grafting (CABG). Pericardial fat was collected intraoperatively and examined for capillary sprouting capacity using an established ex vivo Matrigel-based methodology quantified for growth over seven days. Angiogenic capacity was significantly blunted (2.5-fold, p&lt;0.001) in subjects with CAD (age 59±2 years, BMI 32±1 kg/m 2 , LDL-C 92±45 mg/dl, n=37) compared to age-, BMI-, and LDL-cholesterol matched individuals without angiographic obstructive CAD (age 56±4 years, BMI 32±1 kg/m 2 , LDL-C 110±40 mg/dl, n=14). To garner potential mechanistic insight, we performed transcriptomic analyses using quantitative RT- PCR for select angiogenic regulators. We observed no significant differences in pericardial fat gene expression for pro-angiogenic mediators vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and angiopoietin-1, or anti-angiogenic factors including soluble fms-like tyrosine kinase-1 (sFlt-1) and endostatin. In contrast, mRNA expression of anti-angiogenic thrombospondin-1 (TSP1) was significantly upregulated (2-fold, p&lt;0.05) in CAD subjects compared to the non-CAD group, which was confirmed by protein western-immunoblot analysis. Plasma concentrations of TSP-1 quantified by ELISA were not significantly different between the two groups. In summary, pericardial fat in subjects with CAD may be associated with an anti-angiogenic transcriptome linked to functional defects in vascularization capacity. Local paracrine actions of TSP1 in adipose depots surrounding the heart may play a role in mechanisms of ischemic heart disease.

Development of a population pharmacokinetic/pharmacodynamic model for various oral paclitaxel formulations co-administered with ritonavir and thrombospondin-1 based on data from early phase clinical studies

PurposeOrally administered paclitaxel offers increased patient convenience while providing a method to prolong exposure without long continuous, or repeated, intravenous infusions. The oral bioavailability of paclitaxel is improved through co-administration with ritonavir and application of a suitable pharmaceutical formulation, which addresses the dissolution-limited absorption of paclitaxel. We aimed to characterize the pharmacokinetics of different paclitaxel formulations, co-administered with ritonavir, and to investigate a pharmacodynamic relationship between low-dose metronomic (LDM) treatment with oral paclitaxel and the anti-angiogenic marker thrombospondin-1 (TSP-1).MethodsFifty-eight patients treated with different oral paclitaxel formulations were included for pharmacokinetic analysis. Pharmacodynamic data was available for 36 patients. All population pharmacokinetic/pharmacodynamic modelling was performed using non-linear mixed-effects modelling.ResultsA pharmacokinetic model consisting of gut, liver, central, and peripheral compartments was developed for paclitaxel. The gastrointestinal absorption rate was modelled with a Weibull function. Relative gut bioavailabilities of the tablet and capsule formulations, as fractions of the gut bioavailability of the drinking solution, were estimated to be 0.97 (95%CI: 0.67–1.33) and 0.46 (95%CI: 0.34–0.61), respectively. The pharmacokinetic/pharmacodynamic relationship between paclitaxel and TSP-1 was modelled using a turnover model with paclitaxel plasma concentrations driving an increase in TSP-1 formation rate following an Emax relationship with an EC50 of 284 ng/mL (95%CI: 122–724).ConclusionThe developed pharmacokinetic model adequately described the paclitaxel plasma concentrations for the different oral formulations co-administered with ritonavir. This model, and the established pharmacokinetic/pharmacodynamic relationship with TSP-1, may facilitate future development of oral paclitaxel.

Inhibition of oxidative stress delays senescence and augments functional capacity of endothelial progenitor cells

Ageing is characterised by a progressive loss of vascular endothelial function and integrity. Endothelial progenitor cells (EPCs) play an integral role in endothelial regeneration but are prone to age-dependent changes which may accelerate their senescence and diminish their availability and functionality. Considering these, we firstly investigated the quantity of circulating EPCs in older (73.3 ± 7.2 years) and younger (40.2 ± 14.3 years) healthy volunteers and showed sharp declines in the number of EPCs expressing stemness markers (CD34 + and/or CD133 + ) in older people. These coincided with the decreases in total anti-oxidant capacity (TAC) and concomitant increases in plasma levels of pro-inflammatory cytokine, TNF-α and anti-angiogenic factor, endostatin and thrombospondin-1. The subsequent experimental studies to scrutinise the effect of ageing on molecular and functional properties of outgrowth endothelial cells (OECs), the functional subtype of EPCs, showed that chronological ageing, mimicked by replicative senescence, profoundly impaired proliferation, migration, tubulogenesis, and blood–brain barrier (BBB)-forming capacity of these cells. Similar to those seen in the clinical observational studies, senescent OECs also manifested decreased TAC and increased pro-oxidant NADPH oxidase activity and endostatin level. Suppressing oxidative stress level using structurally and functionally distinct anti-oxidants, namely vitamin C or VAS2870, an NADPH oxidase inhibitor, delayed OEC senescence and restored their tubulogenic and BBB-forming capacities. In conclusion, the enhanced oxidative stress level that develops during physiological ageing may promote EPC senescence and evoke endothelial dysfunction. Effective control of oxidative stress using either compound somewhat delays both phenomena and augments EPC functionality.

Neuronal GPR81 regulates developmental brain angiogenesis and promotes brain recovery after a hypoxic ischemic insult

Perinatal hypoxic/ischemic (HI) brain injury is a major clinical problem with devastating neurodevelopmental outcomes in neonates. During HI brain injury, dysregulated factor production contributes to microvascular impairment. Glycolysis-derived lactate accumulated during ischemia has been proposed to protect against ischemic injury, but its mechanism of action is poorly understood. Herein, we hypothesize that lactate via its G-protein coupled receptor (GPR81) controls postnatal brain angiogenesis and plays a protective role after HI injury. We show that GPR81 is predominantly expressed in neurons of the cerebral cortex and hippocampus. GPR81-null mice displayed a delay in cerebral microvascular development linked to reduced levels of various major angiogenic factors and augmented expression of anti-angiogenic Thrombospondin-1 (TSP-1) in comparison to their WT littermates. Coherently, lactate stimulation induced an increase in growth factors (VEGF, Ang1 and 2, PDGF) and reduced TSP-1 expression in neurons, which contributed to accelerating angiogenesis. HI injury in GPR81-null animals curtailed vascular density and consequently increased infarct size compared to changes seen in WT mice; conversely intracerebroventricular lactate injection increased vascular density and diminished infarct size in WT but not in GPR81-null mice. Collectively, we show that lactate acting via GPR81 participates in developmental brain angiogenesis, and attenuates HI injury by restoring compromised microvasculature.

Time course of capillary regression and an expression balance between vascular endothelial growth factor-A and thrombospondin-1 in the soleus muscle of hindlimb unloaded rats.

Skeletal muscle capillaries regress with disuse; however, information on time-dependent changes in the expression of pro- and anti-angiogenic factors in disused muscle is limited. This study aimed to clarify time-dependent changes in skeletal muscle capillarization, pro-angiogenic vascular endothelial growth factor-A (VEGF-A), and anti-angiogenic thrombospondin-1 (TSP-1) in the soleus muscle of hindlimb unloaded rat. Eight-week-old male Sprague Dawley rats were randomly divided into four groups corresponding to different hindlimb unloading (HU) duration at 0, 1, 2, and 3wk. Muscle atrophy and capillary regression worsened in the soleus muscle with longer periods of HU. The VEGF-A protein expression level was lower at week 1 than at week 0. In addition, the value at week 3 was also lower than those at weeks 0, 1, and 2. The TSP-1 protein expression level was higher at week 1 than that at week 0 but was similar at weeks 2 and 3. Moreover, reactive oxygen species, assessed by dihydroethidium fluorescence intensity on cryosection, were higher at weeks 2 and 3 than that at week 0. Depending on the HU period, VEGF-A and TSP-1 showed different expression patterns. In the early HU phase, TSP-1 may play an important role in capillary regression. However, when HU extends for a longer period, decreased VEGF-A, and/or increased oxidative stress may be more involved in capillary regression.

The BET Protein Inhibitor Apabetalone Rescues Diabetes-Induced Impairment of Angiogenic Response by Epigenetic Regulation of Thrombospondin-1.

Aims: Therapeutic modulation of blood vessel growth holds promise for the prevention of limb ischemia in diabetic (DM) patients with peripheral artery disease (PAD). Epigenetic changes, namely, posttranslational histone modifications, participate in angiogenic response suggesting that chromatin-modifying drugs could be beneficial in this setting. Apabetalone (APA), a selective inhibitor of bromodomain (BRD) and bromodomain and extraterminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating transcriptional programs in different organs. We sought to investigate whether APA affects angiogenic response in diabetes. Results: Compared with vehicle, APA restored tube formation and migration in human aortic endothelial cells (HAECs) exposed to high-glucose (HG) levels. Expression profiling of angiogenesis genes showed that APA prevents HG-induced upregulation of the antiangiogenic molecule thrombospondin-1 (THBS1). ChIP-seq and chromatin immunoprecipitation (ChIP) assays in HG-treated HAECs showed the enrichment of both BRD4 and active marks (H3K27ac) on THBS1 promoter, whereas BRD4 inhibition by APA prevented chromatin accessibility and THBS1 transcription. Mechanistically, we show that THBS1 inhibits angiogenesis by suppressing vascular endothelial growth factor A (VEGFA) signaling, while APA prevents these detrimental changes. In diabetic mice with hind limb ischemia, epigenetic editing by APA restored the THBS1/VEGFA axis, thus improving limb vascularization and perfusion, compared with vehicle-treated animals. Finally, epigenetic regulation of THBS1 by BRD4/H3K27ac was also reported in DM patients with PAD compared with nondiabetic controls. Innovation: This is the first study showing that BET protein inhibition by APA restores angiogenic response in experimental diabetes. Conclusions: Our findings set the stage for preclinical studies and exploratory clinical trials testing APA in diabetic PAD. Antioxid. Redox Signal. 36, 667-684.

Thrombospondin-1 Silencing Improves Lymphocyte Infiltration in Tumors and Response to Anti-PD-1 in Triple-Negative Breast Cancer.

Simple SummaryTriple-negative breast cancer (TNBC) is associated with a poor prognosis, and the development of better therapeutic strategies is required. Herein, we investigated the role of the anti-angiogenic thrombospondin-1 (TSP1) in TNBC. TSP1 expression in tumor biopsies from TNBC patients was associated with a bad prognosis and a weak content of tumor-infiltrating lymphocytes (TILs). In the 4T1 mouse TNBC model, TSP1 knockdown reduced TGF-β activation and enhanced the content of TILs. Moreover, TSP1 knockdown decreased lung metastasis in syngeneic Balb/c immunocompetent mice but not in immunodeficient nude mice. Finally, TSP1 knockdown enhanced anti-PD-1 immunotherapy efficacy. Thus, targeting TSP1 may be considered as a putative therapeutic strategy in TNBC in combination with immunotherapy.Triple-negative breast cancer (TNBC) is notoriously aggressive with a high metastatic potential, and targeted therapies are lacking. Using transcriptomic and histologic analysis of TNBC samples, we found that a high expression of thrombospondin-1 (TSP1), a potent endogenous inhibitor of angiogenesis and an activator of latent transforming growth factor beta (TGF-β), is associated with (i) gene signatures of epithelial–mesenchymal transition and TGF-β signaling, (ii) metastasis and (iii) a reduced survival in TNBC patients. In contrast, in tumors expressing low levels of TSP1, gene signatures of interferon gamma (IFN-γ) signaling and lymphocyte activation were enriched. In TNBC biopsies, TSP1 expression inversely correlated with the CD8+ tumor-infiltrating lymphocytes (TILs) content. In the 4T1 metastatic mouse model of TNBC, TSP1 silencing did not affect primary tumor development but, strikingly, impaired metastasis in immunocompetent but not in immunodeficient nude mice. Moreover, TSP1 knockdown increased tumor vascularization and T lymphocyte infiltration and decreased TGF-β activation in immunocompetent mice. Noteworthy was the finding that TSP1 knockdown increased CD8+ TILs and their programmed cell death 1 (PD-1) expression and sensitized 4T1 tumors to anti-PD-1 therapy. TSP1 inhibition might thus represent an innovative targeted approach to impair TGF-β activation and breast cancer cell metastasis and improve lymphocyte infiltration in tumors, and immunotherapy efficacy in TNBC.

Abstract 5: Experimentally-driven mathematical model of tumor angiogenesis mediated by multiple angiogenic factors

Abstract Tumor angiogenesis is regulated by multiple pro- and anti-angiogenic factors. Anti-angiogenic drugs target the interconnected network of these angiogenic factors to inhibit neovascularization and tumor growth. However, current anti-angiogenic therapeutics targeting a single angiogenic factor show limited clinical success, prompting the development of combination anti-angiogenic therapy targeting multiple angiogenic factors simultaneously. In cases where anti-angiogenic therapy may be effective, tumors display a wide range of responses, which is not fully understood. In this work, we use an integrative in vitro imaging and mathematical modeling approach to investigate how tumor angiogenesis is systematically regulated by multiple angiogenic factors and to study the effects of anti-angiogenic therapy. We particularly focus on two pro-angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2), and two anti-angiogenic factors, thrombospondin-1 (TSP1) and platelet factor 4 (PF4). In our in vitro experimental setting human endothelial cells are cultured in a microfluidic organ-on-chip chip platform exposed to angiogenic factors or in the presence of cancer cells to mimic the tumor microenvironment. The cellular responses induced by various concentrations of angiogenic factors are measured through a high content screening (HCS) confocal imaging system to quantify the cell counts, morphological features and vessel characteristics. These data are used to construct a novel mathematical model to capture how the angiogenic factors mediate cross-talk between tumor cells and endothelial cells. By fitting the model to the experimental measurements, we are able to predict the temporal dynamics of cell numbers and concentrations of the angiogenic factors. Using the model, we generate insights into the effects of angiogenic factors and various anti-angiogenic treatments on tumor cells and endothelial cells. Excitingly, the model can also be applied to investigate how the effect of anti-angiogenic therapy is influenced by inter-tumor heterogeneity. For example, we identified that tumors with lower tumor cell growth rate and higher carrying capacity have a stronger response to anti-VEGF treatment, which indicates that variation in tumor cell growth rates can be a main reason for the observed heterogenous response to anti-VEGF therapy. In addition, we investigate a novel hypothesis regarding synergy between anti-angiogenic and chemotherapeutic agents with the model. Our simulated results suggest a new mechanism in which the chemotherapeutic agent enhances anti-angiogenic therapy simply through reducing the tumor cell growth rate. Overall, this work generates novel insights into the function of multiple angiogenic factors in tumor angiogenesis, providing a tool that can be further used to test and optimize anticancer therapy. Citation Format: Ding Li, Danielle D. Hixon, Shannon M. Mumenthaler, Stacey D. Finley. Experimentally-driven mathematical model of tumor angiogenesis mediated by multiple angiogenic factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 5.

Mild hyperbaric oxygen exposure attenuates rarefaction of capillary vessels in streptozotocin-induced diabetic soleus muscle in rats.

We examined the effects of mild hyperbaric oxygen (mHBO) exposure on capillary rarefaction in skeletal muscles of rats with diabetes. Streptozotocin (100 mg/kg) was administered to male Wistar rats via the tail vein to prepare a diabetic model. These rats were divided into 2 groups: the group with mHBO exposure (1.25 atmospheres absolute (ATA) with 36% oxygen; 3 h/day) and the group without mHBO exposure. Age-matched rats were used as the control group. Eight weeks later, the soleus of the rats was removed and then analyzed. With the onset of diabetes mellitus, capillary number, diameter, and volume in the soleus of the rats with diabetes decreased compared with those of the rats in the control group. In addition, increased anti-angiogenic thrombospondin-1 (TSP-1) and decreased pro-angiogenic murine double minute 2 (MDM-2) protein expressions were observed in the rats with diabetes. Alternatively, mHBO exposure attenuated the decrease in capillary diameter and volume in skeletal muscles of rats with diabetes, suppressed the overexpression of TSP-1, and restored the MDM-2 expression. These results indicate the exposure of mHBO partially attenuates capillary rarefaction in diabetic soleus muscle.

Thrombospondin1 as a potential therapeutic target for human nonalcoholic fatty liver disease.

Thrombospondin1 as a potential therapeutic target for human nonalcoholic fatty liver disease.

Collagen IX deficiency leads to premature vascularization and ossification of murine femoral heads through an imbalance of pro- and antiangiogenic factors

The vascular invasion of cartilage is an essential process in the endochondral ossification of long bones. In contrast, vascularization of articular cartilage constitutes a pathological mechanism in the development of osteoarthritis. Polymorphisms of Col9a1 have been described as risk factors for hip osteoarthritis (OA) and the loss of collagen IX is known to lead to premature OA of the hip joint in mice but the underlying mechanism is so far unknown. To understand the contribution of collagen IX to OA development in the hip joint, we analyzed the early development of murine Col9a1-/- femoral heads between newborn stage and 16 weeks of age. We found significantly accelerated ossification of the femoral heads in the absence of collagen IX as well as premature vascular and osteoclast invasion, even though hypertrophic differentiation was delayed. The loss of collagen IX led to anatomically altered femoral heads lacking the epiphyseal tubercle. Interestingly, this region was found to contain highest levels of the antiangiogenic protein thrombospondin-1 (TSP-1). Hence, TSP-1 levels were strongly reduced in the Col9a1-/- femoral heads. In addition, antiangiogenic matrilin-1 was found to be decreased, while proangiogenic active MMP-9 levels were increased in the collagen IX deficient mice compared to wildtype controls. We conclude that collagen IX protects against premature vascularization and cartilage to bone transition in femoral heads by increasing the levels of antiangiogenic TSP-1 and matrilin-1 and decreasing the levels of proangiogenic active MMP-9.

Mesenchymal Stem/Stromal Cells from the Placentae of Growth Restricted Pregnancies Are Poor Stimulators of Angiogenesis.

The extensively branched vascular network within the placenta is vital for materno-fetal exchange, and inadequate development of this network is implicated in the pregnancy disorder fetal growth restriction (FGR), where babies are born pathologically small. Placental mesenchymal stem/stromal cells (pMSCs) and placental macrophages both reside in close proximity to blood vessels within the placenta, where they are thought to promote angiogenesis via paracrine mechanisms. However, the relationship between pMSCs, macrophages and placental vascular development has not yet been examined. We aimed to determine if inadequate paracrine stimulation of placental vascular development by pMSCs and macrophages during pregnancy may contribute to the inadequate vascularisation seen in FGR. Media conditioned by MSCs from FGR placentae significantly inhibited endothelial tube formation, compared to conditioned media derived from normal pMSCs. Similarly, macrophages exposed to media conditioned by FGR pMSCs were less able to stimulate endothelial tube formation in comparison to macrophages exposed to media conditioned by normal pMSCs. While MSCs from normal placentae produce a combination of angiogenic and anti-angiogenic cytokines, there were no significant differences in the secretion of the anti-angiogenic cytokines thrombospondin-1, insulin growth factor binding protein-4, or decorin between normal and FGR pMSCs that could explain how FGR pMSCs inhibited endothelial tube formation. Together, these data suggest a dysregulation in the secretion of paracrine factors by pMSCs in FGR placentae. These findings illustrate how cross talk between pro-angiogenic cell types in the placenta may be crucial for adequate angiogenesis.

Treatment with a Combination of Metformin and 2-Deoxyglucose Upregulates Thrombospondin-1 in Microvascular Endothelial Cells: Implications in Anti-Angiogenic Cancer Therapy.

Metformin, the most widely used anti-diabetic drug, also exhibits anti-cancer properties; however, the true potential of metformin as an anticancer drug remains largely unknown. In this study using mouse microvascular endothelial cells (MMECs), we investigated the effects of metformin alone or in combination with the glycolytic inhibitor, 2-deoxyglucose (2DG), on angiogenesis-a process known to be an integral part of tumor growth, cancer cell survival and metastasis. MMECs were exposed to 2DG (1–10 mM) for 48 h in the absence or presence of metformin (2 mM). The status of angiogenic and anti-angiogenic marker proteins, proteins of the mTOR pathway and cell-cycle-related proteins were quantified by Western blot analysis. Assays for cell proliferation, migration and tubulogenesis were also performed. We observed robust up-regulation of anti-angiogenic thrombospondin-1 (TSP1) and increased TSP1-CD36 co-localization with a marked decrease in the levels of phosphorylated vascular endothelial growth factor receptor-2 (pVEGFR2; Y1175) in 2DG (5 mM) exposed cells treated with metformin (2 mM). Additionally, treatment with metformin and 2DG (5 mM) inhibited the Akt/mTOR pathway and down-regulated the cell-cycle-related proteins such as p-cyclin B1 (S147) and cyclins D1 and D2 when compared to cells that were treated with either 2DG or metformin alone. Treatment with a combination of 2DG (5 mM) and metformin (2 mM) also significantly decreased cell proliferation, migration and tubulogenic capacity when compared to cells that were treated with either 2DG or metformin alone. The up-regulation of TSP1, inhibition of cell proliferation, migration and tubulogenesis provides support to the argument that the combination of metformin and 2DG may prove to be an appropriate anti-proliferative and anti-angiogenic therapeutic strategy for the treatment of some cancers.

Effects of Obesity and Acute Resistance Exercise on Skeletal Muscle Intercellular Communication Pathways

Obesity (OB) disrupts cellular communication consistent with lower skeletal muscle capillarization. Exosomes, small microvesicles, transport and deliver mRNA, miRNA, and proteins in an endocrine manner and are released by muscle during aerobic exercise. The effects of resistance exercise (REx) on exosome biogenesis is unknown. PURPOSE: Investigate if resistance exercise increases skeletal muscle exosome biogenesis pathways and if this response is impaired in obesity. METHODS: Lean (LN) and obese (OB) (n=8/group) sedentary men and women performed 3 sets of 8-12 repetitions/set of acute, single leg knee extension resistance exercise at 80% of 1-RM. Vastus lateralis biopsies were obtained at rest and at 15 min, and 3 hr post-exercise. Muscle mRNA, protein expression, fiber typing, and capillary staining were measured. RESULTS: The gene expression of the exosome biogenesis components hepatocyte growth factor-regulated tyrosine kinase (HGS) and vacuolar protein sorting mutant (VPS4a) were lower in OB than LN at rest (~25%) and at 15 min post- (~20%), but not 3 hr post-exercise. Expression of exosome surface markers apoptotic linked gene-2 interacting protein X (Alix) was lower (OB ~35% and LN ~20% 15min post-exercise) and tumor susceptibility gene-101 (TSG-101) was higher (OB ~50% and LN ~40% 3hr post-exercise) in response to REx in both groups. Acute resistance exercise increased vascular endothelial growth factor (VEGF) mRNA similarly in LN and OB. Interestingly, anti-angiogenic thrombospondin-1 (TSP-1) mRNA was increased by acute REx only in OB (~230% 3hr post-exercise). miR-130a (angiogenesis), miR-206 (myoblast to myotube differentiation) and miR-503 (repressor of cell proliferation) were increased in OB at rest and following exercise. Type II fiber size was greater and capillary density was lower in OB. CONCLUSION: Obesity alters skeletal muscle exosome biogenesis, angiogenic, and muscle differentiation pathways possibly contributing to greater muscle fiber size and lower muscle capillarization. Resistance exercise alters skeletal muscle exosome marker expression similarly in both lean and obese.

Hypercholesterolemia-induced increase in plasma oxidized LDL abrogated pro angiogenic response in kidney grafts

BackgroundRenal transplantation is increasingly associated with the presence of comorbidity factors such as dyslipidemia which could influence the graft outcome. We hypothesized that hypercholesterolemia could affect vascular repair processes and promote post-transplant renal vascular remodeling through the over-expression of the anti-angiogenic thrombospondin-1 interacting with vascular endothelial growth factor-A levels.MethodsWe tested this hypothesis in vitro, in vivo and in a human cohort using (1) endothelial cells; (2) kidney auto-transplanted pig subjected (n = 5) or not (n = 6) to a diet enriched in cholesterol and (3) a renal transplanted patient cohort (16 patients).ResultsCells exposed to oxidized LDL showed reduced proliferation and an increased expression of thrombospondin-1. In pigs, 3 months after transplantation of kidney grafts, we observed a deregulation of the hypoxia inducible factor 1a—vascular endothelial growth factor-A axis induced in cholesterol-enriched diet animals concomitant with an overexpression of thrombospondin-1 and a decrease in cortical microvessel density promoting vascular remodeling. In patients, hypercholesterolemia was associated with decreased vascular endothelial growth factor-A plasma levels during early follow up after renal transplantation and increased chronic graft dysfunction.ConclusionsThese results support a potential mechanism through which a high fat-diet impedes vascular repair in kidney graft and suggest the value of controlling cholesterolemia in recipient even at the early stage of renal transplantation.

Hyperglycaemia correlates with skeletal muscle capillary regression and is associated with alterations in the murine double minute-2/forkhead box O1/thrombospondin-1 pathway in type 1 diabetic BioBreeding rats.

Type 1 diabetes can have deleterious effects on skeletal muscle and its microvasculature. Our laboratory has recently identified murine double minute-2 as a master regulator of muscle microvasculature by controlling expression levels of two key molecular actors of the angio-adaptive process: the pro-angiogenic vascular endothelial growth factor-A and the anti-angiogenic thrombospondin-1. Here, we show for the first time that in the soleus and plantaris muscles of the diabetes-prone BioBreeding rats, a rodent model of autoimmune type 1 diabetes, murine double minute-2 protein levels are significantly decreased, coinciding with elevated protein levels of thrombospondin-1 and its transcription factor forkhead box O1. Significant capillary regression was observed to similar extent in soleus and plantaris muscles of type 1 diabetic rats. Elevated blood glucose levels were correlated with the loss of capillaries, the reduction in murine double minute-2 expression and with the elevations in thrombospondin-1. Vascular endothelial growth factor-A protein levels were unaltered or even increased in diabetic animals, yet type 1 diabetic animals had less vascular endothelial growth factor receptor-2 abundance. The vascular endothelial growth factor-A/thrombospondin-1 ratio, a good indicator of skeletal muscle angio-adaptive environment, was decreased in type 1 diabetic muscle. Our results suggest that the murine double minute-2-forkhead box O1-thrombospondin-1 pathway plays an important role in angio-regulation of the skeletal muscle in the pathophysiological context of type 1 diabetes.

Platelet microparticle delivered microRNA-Let-7a promotes the angiogenic switch

Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3’UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.

Alternative transcription of a shorter, non-anti-angiogenic thrombospondin-2 variant in cancer-associated blood vessels

Thrombospondin-2 (TSP2) is an anti-angiogenic matricellular protein that inhibits tumor growth and angiogenesis. Tumor-associated blood vascular endothelial cells (BECs) were isolated from human invasive bladder cancers and from matched normal bladder tissue by immuno-laser capture microdissection. Exon expression profiling analyses revealed a particularly high expression of a short TSP2 transcript containing only the last 9 (3′) exons of the full-length TSP2 transcript. Using 5′ and 3′ RACE (rapid amplification of cDNA ends) and Sanger sequencing, we confirmed the existence of the shorter transcript of TSP2 (sTSP2) and determined its sequence which completely lacked the anti-angiogenic thrombospondin type 1 repeats domain. The largest open reading frame predicted within the transcript comprises 209 amino acids and matches almost completely the C-terminal lectin domain of full-length TSP2. We produced recombinant sTSP2 and found that unlike the full-length TSP2, sTSP2 did not inhibit vascular endothelial growth factor-A-induced proliferation of cultured human BECs, but in contrast when combined with TSP2 blocked the inhibitory effects of TSP2 on BEC proliferation. In vivo studies with stably transfected A431 squamous cell carcinoma cells revealed that full-length TSP2, but not sTSP2, inhibited tumor growth and angiogenesis. This study reveals that the transcriptional program of tumor stromal cells can change to transcribe a new version of an endogenous angiogenesis inhibitor that has lost its anti-angiogenic activity.

Integration of pro- and anti-angiogenic signals by endothelial cells.

Angiogenesis or neovascularization is a complex multi-step physiological process that occurs throughout life both in normal tissues and in disease. It is tightly regulated by the balance between pro-angiogenic and anti-angiogenic factors. The angiogenic switch has been identified as the key step during tumor progression in which the balance between pro-angiogenic and anti-angiogenic factors leans toward pro-angiogenic stimuli promoting the progression of tumors from dormancy to dysplasia and ultimately malignancy. This event can be described as either the outcome of a genetic event occurring in cancer cells themselves, or the positive and negative cross-talk between tumor-associated endothelial cells and other cellular components of the tumor microenvironment. In recent years, the mechanisms underlying the angiogenic switch have been extensively investigated in particular to identify therapeutic targets that can lead to development of effective therapies. In this review, we will discuss the current findings on the regulatory pathways in endothelial cells that are involved in the angiogenic switch with an emphasis on the role of anti-angiogenic protein, thrombospondin-1 (TSP-1) and pro-angiogenic factor, vascular endothelial growth factor (VEGF).