Tongrong He

Transplantation of Circulating Endothelial Progenitor Cells Restores Endothelial Function of Denuded Rabbit Carotid Arteries

Background and Purpose— Circulating endothelial progenitor cells (EPCs) play an important role in repair of injured vascular endothelium and neovascularization. The present study was designed to determine the effect of EPCs transplantation on the regeneration of endothelium and recovery of endothelial function in denuded carotid arteries. Methods— Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium for 7 days, yielding EPCs. A rabbit model of common carotid artery denudation by passage of a deflated balloon catheter was used to evaluate the effects of EPCs on endothelial regeneration and vasomotor function. Immediately after denudation, autologous EPCs (105 cells in 200 μL saline) or 200 μL saline alone (control) were administered into the lumen of injured artery. Results— Four weeks after transplantation, fluorescence-labeled colonies of EPCs were found in the vessel wall. Local transplantation of EPCs as compared with saline administration accelerated endothelialization and significantly improved endothelium-dependent relaxation when assessed 4 weeks after denudation (n=4 to 5, P<0.05). Transplantation of EPCs did not affect vasomotor function of arterial smooth muscle cells. Protein array analysis of conditioned media obtained from cultured EPCs demonstrated the ability of these cells to produce and release a number of proangiogenic cytokines. Conclusions— We conclude that local delivery of cultured circulating EPCs into the lumen of denuded carotid arteries accelerates endothelialization and improves endothelial function. Paracrine effects of EPCs may contribute to regenerative properties of EPCs.

Angiogenic Function of Prostacyclin Biosynthesis in Human Endothelial Progenitor Cells

The role of prostaglandin production in the control of regenerative function of endothelial progenitor cells (EPCs) has not been studied. We hypothesized that activation of cyclooxygenase (COX) enzymatic activity and the subsequent production of prostacyclin (PGI2) is an important mechanism responsible for the regenerative function of EPCs. In the present study, we detected high levels of COX-1 protein expression and PGI2 biosynthesis in human EPCs outgrown from blood mononuclear cells. Expression of COX-2 protein was almost undetectable under basal conditions but significantly elevated after treatment with tumor necrosis factor-&agr;. Condition medium derived from EPCs hyperpolarized human coronary artery smooth muscle cells, similar to the effect of the PGI2 analog iloprost. The proliferation and in vitro tube formation by EPCs were inhibited by the COX inhibitor indomethacin or by genetic inactivation of COX-1 or PGI2 synthase with small interfering (si)RNA. Impaired tube formation and cell proliferation induced by inactivation of COX-1 were rescued by the treatment with iloprost or the selective peroxisome proliferator–activated receptor (PPAR)&dgr; agonist GW501516 but not by the selective PGI2 receptor agonist cicaprost. Downregulation of PPAR&dgr; by siRNA also reduced angiogenic capacity of EPCs. Iloprost failed to reverse PPAR&dgr; siRNA-induced impairment of angiogenesis. Furthermore, transfection of PGI2 synthase siRNA, COX-1 siRNA, or PPAR&dgr; siRNA into EPCs decreased the capillary formation in vivo after transplantation of human EPCs into the nude mice. These results suggest that activation of COX-1/PGI2/PPAR&dgr; pathway is an important mechanism underlying proangiogenic function of EPCs.

Interleukin-1β augments angiogenic responses of murine endothelial progenitor cells in vitro

Endothelial progenitor cells (EPCs) may provide novel opportunities for therapeutic angiogenesis after ischemic diseases. However, it is unclear how the angiogenic potential of EPCs might be affected by an inflammatory environment. We examine how the potent cytokine interleukin-1β (IL-1β) affects angiovasculogenic responses in EPCs in culture. Mononuclear cells isolated from mouse spleen were plated on fibronectin-coated wells and grown in EGM-2 MV media. Endothelial progenitor cells were phenotyped using multiple markers (UEA-Lectin, ac-LDL, CD133, CD34, vWillebrand Factor, Flk-1) and to identify the IL-1 Receptor-I. We quantified cell and colony counts and performed MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) and Matrigel assays, in vitro, under control and IL-1β (10 ng/mL) conditions. Endothelial progenitor cells exposed to IL-1β increased in the number of cells and colonies compared with untreated cells, without any effect on cell metabolic integrity. Furthermore, IL-1β treatment augmented EPC angiogenic function, significantly increasing the number of vessel-like structures in the Matrigel assay. An early phosphorylation of ERK1/2 occurred after IL-1β stimulation, and this pathway was inhibited if IL-1 Receptor-I was blocked. Our results suggest that IL-1β is a potent stimulator of in vitro angiogenesis through ERK signaling in mouse EPCs. Further studies are warranted to assess how interactions between proinflammatory environments and EPC responses may be leveraged to enhance therapeutic angiogenesis.

Activation of Peroxisome Proliferator-Activated Receptor–δ Enhances Regenerative Capacity of Human Endothelial Progenitor Cells by Stimulating Biosynthesis of Tetrahydrobiopterin

The mechanisms underlying the regenerative capacity of endothelial progenitor cells (EPCs) are not fully understood. We hypothesized that biosynthesis of tetrahydrobiopterin is an important mechanism responsible for the stimulatory effects of peroxisome proliferator-activated receptor–&dgr; (PPAR&dgr;) activation on regenerative function of human EPCs. Treatment of human EPCs with a selective PPAR&dgr; agonist GW501516 for 24 hours increased the levels of mRNA, protein, and enzymatic activity of GTP cyclohydrolase I (GTPCH I), as well as the production of tetrahydrobiopterin. The effects of GW501516 were mediated by suppression of PTEN expression, thereby increasing phosphorylation of AKT. The AKT signaling also mediated GW501516-induced phosphorylation of endothelial NO synthase. In addition, activation of PPAR&dgr; significantly enhanced proliferation of EPCs. This effect was abolished by the GTPCH I inhibitor, 2,4-diamino-6-hydroxypyrimidine, or genetic inactivation of GTPCH I with small interfering RNA but not by inhibition of endothelial NO synthase with NG-nitro-L-arginine methyl ester. Supplementation of NO did not reverse 2,4-diamino-6-hydroxypyrimidine-inhibited 5-bromodeoxyuridine incorporation. Furthermore, transplantation of human EPCs stimulated re-endothelialization in a mouse model of carotid artery injury. Pretreatment of EPCs with GW501516 significantly enhanced the ability of transplanted EPCs to repair denuded endothelium. GTPCH I-small interfering RNA transfection significantly inhibited in vivo regenerative capacity of EPCs stimulated with GW501516. Thus, in human EPCs, activation of PPAR&dgr; stimulates expression and activity of GTPCH I and biosynthesis of tetrahydrobiopterin via PTEN-AKT signaling pathway. This effect enhances the regenerative function of EPCs.

Brain-derived neurotrophic factor increases expression of MnSOD in human circulating angiogenic cells.

Existing evidence suggests that brain-derived neurotrophic factor (BDNF) promotes survival and proliferation of endothelial cells, stimulates mobilization of hematopoietic progenitors, and induces angiogenesis in ischemic tissues. However, the mechanisms underlying vascular protective function of BDNF are poorly understood. We hypothesized that BDNF increases antioxidant capacity of circulating angiogenic cells. Human mononuclear cells were isolated from peripheral blood of 30 healthy male volunteers (48±2 years old), and cultured in endothelial growth medium-2 for 4-5 days. The attached cells (so called early endothelial progenitor cells [early EPCs], or circulating angiogenic cells) expressed BDNF receptors, tropomyosin-related kinase B and p75 neurotrophin receptor. Treatment of early EPCs with recombinant human BDNF for 24 h significantly increased manganese superoxide dismutase (MnSOD) expression, but had no effect on expression of other antioxidant enzymes including copper zinc SOD (CuZnSOD), catalase, and glutathione peroxidase-1. BDNF stimulated phosphorylation of IκB kinase (IKK)α/β and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK); however it did not activate p38, Erk, or AKT. Treatment with nuclear factor κB inhibitor, PDTC, or JNK inhibitor, SP600125, attenuated BDNF-augmented MnSOD protein expression. BDNF treatment inhibited apoptosis induced by a superoxide anion generator LY83583, and serum starvation-induced cell detachment. These findings suggest that BDNF protects EPCs by increasing expression of MnSOD thereby enhancing their antioxidant capacity.

Activation of PPARδ prevents endothelial dysfunction induced by overexpression of amyloid-β precursor protein

AIMS Existing evidence suggests that amyloid-β precursor protein (APP) causes endothelial dysfunction and contributes to pathogenesis of atherosclerosis. In the present study, experiments were designed to: (1) determine the mechanisms underlying endothelial dysfunction and (2) define the effects of peroxisome proliferator-activated receptor delta (PPARδ) ligand on endothelial function in transgenic Tg2576 mice overexpressing mutated human APP. METHODS AND RESULTS Confocal microscopy and western blot analyses of wild-type mice aortas provided evidence that APP protein is mainly present in endothelial cells. Overexpression of APP significantly impaired endothelium-dependent relaxations to acetylcholine and phosphorylation of endothelial nitric oxide synthase at Ser(1177) in aortas. HPLC analysis revealed that tetrahydrobiopterin (BH(4)) levels were reduced in Tg2576 mice aortas. This was caused by increased oxidation of BH(4) and reduced expression and activity of GTP-cyclohydrolase I. Furthermore, gp91phox protein expression and superoxide anion production were increased in aortas of Tg2576 mice. This augmented superoxide formation was completely prevented by the NADPH oxidase inhibitor VAS2870. Expression of copper-/zinc-superoxide dismutase (Cu/ZnSOD) and extracellular SOD was downregulated. Treatment with PPARδ ligand GW501516 (2 mg/kg/day) for 14 days significantly increased BH(4) bioavailability and improved endothelium-dependent relaxations in Tg2576 mice aortas. GW501516 also normalized protein expression of gp91(phox) and SODs, thereby reducing production of superoxide anion in the aortas. CONCLUSION Our results suggest that in APP transgenic mice loss of nitric oxide and increased oxidative stress are the major causes of endothelial dysfunction. The vascular protective effects of GW501516 in Tg2576 mice appear to be critically dependent on prevention of superoxide anion production.

Muscle-Specific F-Box Only Proteins Facilitate BK Channel β 1 Subunit Downregulation in Vascular Smooth Muscle Cells of Diabetes Mellitus

Rationale: Activity of the large conductance Ca2+-activated K+ (BK) channels is profoundly modulated by its &bgr;1 subunit (BK-&bgr;1). However, BK-&bgr;1 expression is downregulated in diabetic vessels. The ubiquitin–proteasome system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-&bgr;1 downregulation in diabetic vessels is unknown. Objective: We hypothesize that UPS facilitates vascular BK-&bgr;1 degradation in diabetes. Methods and Results: Using patch clamp and molecular biological approaches, we found that BK-&bgr;1–mediated channel activation and BK-&bgr;1 protein expression were reduced in aortas of streptozotocin-induced diabetic rats and in human coronary arterial smooth muscle cells (CASMCs) cultured in high glucose. This was accompanied by upregulation of F-box only protein (FBXO)-9 and FBXO-32 (atrogin-1), the key components of the Skp1-Cullin-F-box (SCF) type ubiquitin ligase complex. BK-&bgr;1 expression was suppressed by the FBXO activator doxorubicin but enhanced by FBXO-9 small interfering RNA or by the proteasome inhibitor MG-132. Cotransfection of atrogin-1 in HEK293 cells significantly reduced Flag-hSlo-&bgr;1 expression by 2.16-fold, compared with expression of Flag-hSlo-&bgr;1V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo-&bgr;1 was increased by 1.91-fold, compared with that of hSlo-&bgr;1V146A, whereas cotransfection with atrogin-1&Dgr;F (a nonfunctional mutant without the F-box motif) had no effect. Moreover, inhibition of Akt signaling attenuated the phosphorylation of forkhead box O transcription factor (FOXO)-3a and enhanced atrogin-1 expression, which in turn suppressed BK-&bgr;1 protein levels in human CASMCs. Conclusions: Downregulation of vascular BK-&bgr;1 expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-&bgr;1 degradation.

Vascular effects of prostacyclin: does activation of PPARδ play a role?

Prostacyclin (PGI(2)) is a potent vasodilator that exerts multiple vasoprotective effects in the cardiovascular system. The effects of PGI(2) are mediated by activation of the cell membrane G-protein-coupled PGI(2) receptor (IP receptor). More recently, however, it has been suggested that PGI(2) might also serve as an endogenous ligand and activator of nuclear peroxisome proliferator-activated receptorδ (PPARδ). Consistent with this concept, studies designed to define pharmacological properties of stable PGI(2) analogs revealed that beneficial effects of these compounds appear to be mediated, in part, by activation of PPARδ. This review discusses emerging evidence regarding the contribution of PPARδ activation to vasoprotective and regenerative functions of PGI(2) and stable analogs of PGI(2).

Uncoupling of endothelial nitric oxide synthase in cerebral vasculature of Tg2576 mice

In this study, we tested the hypothesis that reduced bioavailability of tetrahydrobiopterin (BH4) is a major mechanism responsible for pathogenesis of endothelial dysfunction in cerebral microvessels of transgenic mice expressing the Swedish double mutation of human amyloid precursor protein (APP) (Tg2576 mice). Endothelial nitric oxide synthase (eNOS) protein expression was significantly increased in cerebral vasculature of Tg2576 mice. In contrast, bioavailability of BH4 was significantly reduced (p < 0.05). Moreover, superoxide anion production was increased in cerebral microvessels of Tg2576 mice (p < 0.05). Incubation with NOS inhibitor, Nω‐nitro‐L‐arginine methyl ester, decreased superoxide anion indicating that uncoupled eNOS is most likely the source of superoxide anion. Increasing BH4 bioavailability either exogenously by BH4 supplementation or endogenously by treatment with the selective peroxisome proliferator‐activated receptor – delta activator GW501516 (2 mg/kg/day, 14 days) attenuated eNOS uncoupling and decreased superoxide anion production in cerebral microvessels of Tg2576 mice (p < 0.05). Treatment with GW501516 restored the biological activity of endothelial nitric oxide in cerebral microvessels of Tg2576 mice, as indicated by the increased nitrite/nitrate content and 3,5‐cyclic guanosine monophosphate levels (p < 0.05). Our studies indicate that sub‐optimal BH4 bioavailability in cerebral vasculature is an important contributor to oxidant stress and endothelial dysfunction in Tg2576 mouse model of Alzheimers disease.

PPARδ agonist GW501516 prevents uncoupling of endothelial nitric oxide synthase in cerebral microvessels of hph-1 mice

Peroxisome proliferator-activated receptor delta (PPARδ) is ubiquitously expressed in the vasculature, including cerebral circulation. The role of PPARδ in metabolism of tetrahydrobiopterin (BH₄) has not been studied in the cerebral microvasculature. In the present study, the effects of PPARδ agonist GW501516 on uncoupling of endothelial nitric oxide synthase (eNOS) were determined in cerebral microvessels of BH₄-deficient hph-1 mice. Wild-type (B6CBA) and hph-1 mice were orally gavaged with a selective PPARδ activator, GW501516 (2 mg/kg/day) for 14 days, and thereafter, cerebral microvessels were isolated and studied. Treatment of hph-1 mice with GW501516 significantly reduced oxidation of BH₄ and increased the ratio of BH₄ to 7,8-BH₂ (P<0.05, n=6-9). Attenuation of L-NAME-inhibitable superoxide anion levels by GW501516 demonstrated that activation of PPARδ might prevent uncoupling of endothelial nitric oxide synthase (eNOS, P<0.05, n=6-9). Western blotting studies demonstrated that GW501516 selectively increased the endothelial expressions of CuZn superoxide dismutase (P<0.05, n=6-9) and catalase (P<0.05, n=6-8). PPARδ activation increased the total nitrite and nitrate (NO₂+NO₃) content in cerebral microvessels (P<0.05, n=6). Obtained results suggest that in vivo activation of PPARδ prevents eNOS uncoupling, restores bioavailability of NO and may help preserve endothelial function in the BH₄-deficient cerebral circulation.