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1.
Am J Physiol Heart Circ Physiol ; 312(4): H711-H720, 2017 Apr 01.
Article in English | MEDLINE | ID: mdl-28188215

ABSTRACT

Endothelial cystathionine γ-lyase (CSEγ) contributes to cardiovascular homeostasis, mainly through production of H2S. However, the molecular mechanisms that control CSEγ gene expression in the endothelium during cardiovascular diseases are unclear. The aim of the current study is to determine the role of specific histone deacetylases (HDACs) in the regulation of endothelial CSEγ. Reduced CSEγ mRNA expression and protein abundance were observed in human aortic endothelial cells (HAEC) exposed to oxidized LDL (OxLDL) and in aortas from atherogenic apolipoprotein E knockout (ApoE-/-) mice fed a high-fat diet compared with controls. Intact murine aortic rings exposed to OxLDL (50 µg/ml) for 24 h exhibited impaired endothelium-dependent vasorelaxation that was blocked by CSEγ overexpression or the H2S donor NaHS. CSEγ expression was upregulated by pan-HDAC inhibitors and by class II-specific HDAC inhibitors, but not by other class-specific inhibitors. The HDAC6 selective inhibitor tubacin and HDAC6-specific siRNA increased CSEγ expression and blocked OxLDL-mediated reductions in endothelial CSEγ expression and CSEγ promoter activity, indicating that HDAC6 is a specific regulator of CSEγ expression. Consistent with this finding, HDAC6 mRNA, protein expression, and activity were upregulated in OxLDL-exposed HAEC, but not in human aortic smooth muscle cells. HDAC6 protein levels in aortas from high-fat diet-fed ApoE-/- mice were comparable to those in controls, whereas HDAC6 activity was robustly upregulated. Together, our findings indicate that HDAC6 is upregulated by atherogenic stimuli via posttranslational modifications and is a critical regulator of CSEγ expression in vascular endothelium. Inhibition of HDAC6 activity may improve endothelial function and prevent or reverse the development of atherosclerosis.NEW & NOTEWORTHY Oxidative injury to endothelial cells by oxidized LDL reduced cystathionine γ-lyase (CSEγ) expression and H2S production, leading to endothelial dysfunction, which was prevented by histone deacetylase 6 (HDAC6) inhibition. Our data suggest HDAC6 as a novel therapeutic target to prevent the development of atherosclerosis.


Subject(s)
Cystathionine gamma-Lyase/metabolism , Endothelium, Vascular/enzymology , Endothelium, Vascular/physiology , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/genetics , Animals , Apolipoproteins E/genetics , Cystathionine gamma-Lyase/biosynthesis , Cystathionine gamma-Lyase/genetics , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelium, Vascular/drug effects , Histone Deacetylase 6 , Humans , Hydrogen Sulfide/metabolism , Hydrogen Sulfide/pharmacology , Lipoproteins, LDL/toxicity , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardial Contraction , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Vasodilation/drug effects , Vasodilation/genetics
2.
J Vis Exp ; (94)2014 Dec 02.
Article in English | MEDLINE | ID: mdl-25489842

ABSTRACT

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to "off-target" effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.


Subject(s)
Disease Models, Animal , Polycystic Kidney Diseases/genetics , Wnt Proteins/genetics , Animals , Animals, Genetically Modified , Gene Knockdown Techniques , Green Fluorescent Proteins/genetics , Oligonucleotides, Antisense/genetics , Polycystic Kidney Diseases/metabolism , Wnt Proteins/deficiency , Wnt Proteins/metabolism , Wnt-5a Protein , Zebrafish
3.
J Hypertens ; 31(10): 1939-43, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23881298

ABSTRACT

Endothelial dysfunction plays a key role in the development and progression of cardiovascular disease. In patients with hypertension, endothelial dysfunction is characterized by a decrease of vasodilator factors release. Recent evidence highlights the involvement of regulatory T cell in the cardiovascular physiology and pathology. An increasing body of data suggest that an imbalance in the immune system triggers inflammation and compromises the cardiovascular homeostasis. In this mini-review, we will highlight the role of immune regulatory T cells in hypertension-induced vascular dysfunction.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Forkhead Transcription Factors/metabolism , Hypertension/immunology , Interleukin-2 Receptor alpha Subunit/metabolism , T-Lymphocytes, Regulatory/immunology , Vascular Diseases/immunology , Animals , Atherosclerosis/complications , Atherosclerosis/immunology , Blood Pressure , Endothelium, Vascular/pathology , Homeostasis , Humans , Hypertension/complications , Immune Tolerance , Inflammation , Mice , Microcirculation
4.
Circulation ; 121(9): 1104-12, 2010 Mar 09.
Article in English | MEDLINE | ID: mdl-20176991

ABSTRACT

BACKGROUND: Little is known about the role of endothelial progenitor cells (EPCs) in atherosclerosis. Accordingly, we performed a series of assessments with hypercholesterolemic (apolipoprotein E-null [ApoE(-/-)]) and wild-type (WT) mice to evaluate how cholesterol influences reendothelialization, atherosclerosis, and EPC function after arterial injury. METHODS AND RESULTS: Unexpectedly, reendothelialization (assessed by resistance to Evans blue staining) and circulating EPC counts (EPC culture assay) were greater in ApoE(-/-) mice than in WT mice, and transplantation of ApoE(-/-) bone marrow in WT mice accelerated endothelial recovery and increased recruitment of bone marrow-derived EPCs to the neoendothelium. Cholesterol concentration-dependently promoted the proliferation (MTS assay) of both ApoE(-/-) and WT EPCs, and the concentration dependence of EPC adhesion (to vitronectin-, collagen type I-, fibronectin-, and laminin-coated plates), migration (modified Boyden chamber assay), and antiapoptotic (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling stain) activity was biphasic. Cholesterol enhanced the messenger RNA expression (quantitative, real-time reverse-transcription polymerase chain reaction) of vascular endothelial growth factor and inhibited Notch1 messenger RNA expression in both ApoE(-/-) and WT EPCs, whereas endothelial nitric oxide synthase messenger RNA expression increased in ApoE(-/-) EPCs and declined in WT EPCs after cholesterol exposure. EPC activity was greater in Notch1(+/-) EPCs than in WT EPCs, and transplantation of Notch1(+/-) bone marrow accelerated endothelial recovery after arterial injury in WT mice. CONCLUSIONS: The results presented here provide novel insights into the role of EPCs during atherosclerosis and suggest that cholesterol and Notch1 may be involved in the regulation of EPC activity.


Subject(s)
Carotid Artery Injuries/pathology , Endothelial Cells/pathology , Hypercholesterolemia/pathology , Mesenchymal Stem Cells/pathology , Receptor, Notch1/physiology , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Atherosclerosis/etiology , Atherosclerosis/genetics , Atherosclerosis/pathology , Bone Marrow Transplantation , Carotid Artery Injuries/complications , Cell Movement , Cholesterol/blood , Gene Expression Regulation , Genotype , Hypercholesterolemia/complications , Hypercholesterolemia/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide Synthase Type III/biosynthesis , Nitric Oxide Synthase Type III/genetics , RNA, Messenger/biosynthesis , Radiation Chimera , Receptor, Notch1/biosynthesis , Receptor, Notch1/deficiency , Receptor, Notch1/genetics , Signal Transduction , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/genetics
5.
Circulation ; 119(5): 699-708, 2009 Feb 10.
Article in English | MEDLINE | ID: mdl-19171856

ABSTRACT

BACKGROUND: Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence suggested that diabetic neuropathy is causally related to impaired angiogenesis and deficient growth factors. Accordingly, we investigated whether diabetic neuropathy could be reversed by local transplantation of EPCs. METHODS AND RESULTS: We found that motor and sensory nerve conduction velocities, blood flow, and capillary density were reduced in sciatic nerves of streptozotocin-induced diabetic mice but recovered to normal levels after hind-limb injection of bone marrow-derived EPCs. Injected EPCs were preferentially and durably engrafted in the sciatic nerves. A portion of engrafted EPCs were uniquely localized in close proximity to vasa nervorum, and a smaller portion of these EPCs were colocalized with endothelial cells. Multiple angiogenic and neurotrophic factors were significantly increased in the EPC-injected nerves. These dual angiogenic and neurotrophic effects of EPCs were confirmed by higher proliferation of Schwann cells and endothelial cells cultured in EPC-conditioned media. CONCLUSIONS: We demonstrate for the first time that bone marrow-derived EPCs could reverse various manifestations of diabetic neuropathy. These therapeutic effects were mediated by direct augmentation of neovascularization in peripheral nerves through long-term and preferential engraftment of EPCs in nerves and particularly vasa nervorum and their paracrine effects. These findings suggest that EPC transplantation could represent an innovative therapeutic option for treating diabetic neuropathy.


Subject(s)
Diabetes Mellitus, Experimental/complications , Diabetic Neuropathies/therapy , Endothelial Cells/cytology , Hematopoietic Stem Cell Transplantation , Sciatic Nerve/blood supply , Animals , Apoptosis , Bone Marrow Cells/cytology , Cell Division , Cells, Cultured , Diabetic Neuropathies/physiopathology , Hematopoietic Stem Cells/cytology , Laser-Doppler Flowmetry , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Neural Conduction , Schwann Cells/cytology , Sciatic Nerve/physiology
6.
J Exp Med ; 204(13): 3257-69, 2007 Dec 24.
Article in English | MEDLINE | ID: mdl-18070934

ABSTRACT

Noncellular differentiation effects have emerged as important mechanisms mediating therapeutic effects of stem or progenitor cell transplantation. Here, we investigated the expression patterns and sources of humoral factors and their regional and systemic biological effects after bone marrow (BM)-derived endothelial progenitor cell (EPC) transplantation into ischemic myocardium. Although most of the transplanted EPCs disappeared within a week, up-regulation of multiple humoral factors was sustained for longer than two weeks, which correlated well with the recovery of cardiac function. To determine the source of the humoral factors, we injected human EPCs into immunodeficient mice. Whereas the expression of human EPC (donor)-derived cytokines rapidly decreased to a nondetectable level within a week, up-regulation of mouse (recipient)-derived cytokines, including factors that could mobilize BM cells, was sustained. Histologically, we observed higher capillary density, a higher proliferation of myocardial cells, a lower cardiomyocyte apoptosis, and reduced infarct size. Furthermore, after EPC transplantation, BM-derived stem or progenitor cells were increased in the peripheral circulation and incorporated into the site of neovascularization and myocardial repair. These data indicate that myocardial EPC transplantation induces humoral effects, which are sustained by host tissues and play a crucial role in repairing myocardial injury.


Subject(s)
Antibody Formation , Cell Transplantation/methods , Endothelial Cells/cytology , Heart/physiology , Myocardial Ischemia/pathology , Stem Cells/cytology , Animals , Apoptosis , Cell Proliferation , Humans , Mice , Mice, Inbred C57BL , Mice, SCID , Models, Biological , Myocardial Infarction/pathology , Stem Cells/metabolism
7.
Circulation ; 114(21): 2261-70, 2006 Nov 21.
Article in English | MEDLINE | ID: mdl-17088460

ABSTRACT

BACKGROUND: Estradiol (E2) modulates the kinetics of circulating endothelial progenitor cells (EPCs) and favorably affects neovascularization after ischemic injury. However, the roles of estrogen receptors alpha (ER alpha) and beta (ER beta) in EPC biology are largely unknown. METHODS AND RESULTS: In response to E2, migration, tube formation, adhesion, and estrogen-responsive element-dependent gene transcription activities were severely impaired in EPCs obtained from ER alpha-knockout mice (ER alphaKO) and moderately impaired in ER betaKO EPCs. The number of ER alphaKO EPCs (42.4+/-1.5; P<0.001) and ER betaKO EPCs (55.4+/-1.8; P=0.03) incorporated into the ischemic border zone was reduced as compared with wild-type (WT) EPCs (72.5+/-1.3). In bone marrow transplantation (BMT) models, the number of mobilized endogenous EPCs in E2-treated mice was significantly reduced in ER alphaKO BMT (WT mice transplanted with ER alphaKO bone marrow) (2.03+/-0.18%; P=0.004 versus WT BMT) and ER betaKO BMT (2.62+/-0.07%; P=0.02 versus WT) compared with WT BMT (2.87+/-0.13%) (WT to WT BMT as control) mice. Capillary density at the border zone of ischemic myocardium also was significantly reduced in ER alphaKO BMT and ER betaKO BMT compared with WT mice (WT BMT, 1718+/-75/mm2; ER alphaKO BMT, 1107+/-48/mm2; ER betaKO BMT, 1567+/-50/mm2). ER alpha mRNA was expressed more abundantly on EPCs compared with ER beta. Moreover, vascular endothelial growth factor was significantly downregulated on ER alphaKO EPCs compared with WT EPCs both in vitro and in vivo. CONCLUSIONS: Both ER alpha and ER beta contribute to E2-mediated EPC activation and tissue incorporation and to preservation of cardiac function after myocardial infarction. ER alpha plays a more prominent role in this process. Moreover, ER alpha contributes to upregulation of vascular endothelial growth factor, revealing possible mechanisms of an effect of E2 on EPC biology. Finally, these data provide additional evidence of the importance of bone marrow-derived EPC phenotype in ischemic tissue repair.


Subject(s)
Bone Marrow Cells/cytology , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/metabolism , Heart/physiopathology , Myocardial Infarction/physiopathology , Myocardial Infarction/surgery , Stem Cell Transplantation , Animals , Cardiotonic Agents/pharmacology , Cell Differentiation , Cells, Cultured , Endothelial Cells/cytology , Estradiol/metabolism , Estradiol/pharmacology , Estrogen Receptor alpha/deficiency , Estrogen Receptor beta/deficiency , Female , Hematopoietic Stem Cell Mobilization , Mice , Mice, Inbred C57BL , Mice, Knockout , Recovery of Function , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/metabolism , Stem Cells/physiology , Up-Regulation , Vascular Endothelial Growth Factor A/metabolism
9.
Proc Natl Acad Sci U S A ; 103(29): 11015-20, 2006 Jul 18.
Article in English | MEDLINE | ID: mdl-16835303

ABSTRACT

The transcription factor E2F1 is known to regulate cell proliferation and has been thought to modulate tumorigenesis via this mechanism alone. Here we show that mice deficient in E2F1 exhibit enhanced angiogenesis. The proangiogenic phenotype in E2F1 deficiency is the result of overproduction of vascular endothelial growth factor (VEGF) and is prevented by VEGF blockade. Under hypoxic conditions, E2F1 down-regulates the expression of VEGF promoter activity by associating with p53 and specifically down-regulating expression of VEGF but not other hypoxia-inducible genes, suggesting a promoter structure context-dependent regulation mechanism. We found that the minimum VEGF promoter mediating transcriptional repression by E2F1 features an E2F1- binding site with four Sp-1 sites in close proximity. These data disclose an unexpected function of endogenous E2F1: regulation of angiogenic activity via p53-dependent transcriptional control of VEGF expression.


Subject(s)
Cell Cycle , E2F1 Transcription Factor/metabolism , Transcription, Genetic/genetics , Tumor Suppressor Protein p53/metabolism , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/genetics , Animals , Cells, Cultured , Down-Regulation , E2F1 Transcription Factor/deficiency , E2F1 Transcription Factor/genetics , Hindlimb/blood supply , Hindlimb/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Tumor Suppressor Protein p53/genetics
10.
Nat Clin Pract Cardiovasc Med ; 3 Suppl 1: S123-8, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16501618

ABSTRACT

Previous studies have shown that local angiogenic gene therapy acts, in part, by recruiting endothelial progenitor cells (EPCs) to ischemic tissue. Recent data indicate that patients with the most severe vascular disease may have insufficient or deficient EPCs and the poorest response to angiogenic therapy. Accordingly, we hypothesized that combining human CD34(+) cell implantation with local vascular endothelial growth factor 2 (phVEGF2) gene therapy might overcome these deficiencies. The addition of VEGF2 to EPC cultures resulted in significant and dose-dependent decreases in EPC apoptosis. Phosphorylated Akt (p-Akt) was increased in VEGF2-treated EPCs. In vivo, myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery in 34 immunodeficient rats. The animals were then randomized to one of four treatment groups: cell therapy alone with human CD34(+) cells; VEGF2 gene therapy alone; combination therapy with CD34(+) cells plus phVEGF2; or CD34(-) cells and 50 microg empty plasmid. Four weeks after MI, animals treated with combination therapy showed improved fractional shortening, increased capillary density, and reduced infarct size compared with the other three groups. Combination therapy was also associated with an increased number of circulating EPCs 1 week after MI. Combined subtherapeutic doses of cell and gene therapy result in a significant therapeutic effect compared to monotherapy. This approach may overcome therapeutic failures (e.g. inability of certain patients to mobilize sufficient EPCs) and may also offer safety advantages by allowing lower dosing strategies.


Subject(s)
Antigens, CD34/analysis , Endothelial Cells/transplantation , Myocardial Infarction/therapy , Vascular Endothelial Growth Factors/metabolism , Animals , Apoptosis , Cells, Cultured , Combined Modality Therapy , Disease Models, Animal , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelial Cells/immunology , Female , Genetic Therapy , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardium/pathology , Rats , Rats, Nude , Transplantation, Autologous , Vascular Endothelial Growth Factors/genetics , Vascular Endothelial Growth Factors/pharmacology
11.
Circ Res ; 98(5): 697-704, 2006 Mar 17.
Article in English | MEDLINE | ID: mdl-16484619

ABSTRACT

Delayed reendothelialization contributes to restenosis after angioplasty and stenting in diabetes. Prior data have shown that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to endothelial recovery after arterial injury. We investigated the hypothesis that the EPC contribution to reendothelialization may be impaired in diabetes, resulting in delayed reendothelialization. Reendothelialization was significantly reduced in diabetic mice compared with nondiabetic mice in a wire-induced carotid denudation model. The EPC contribution to neoendothelium was significantly reduced in Tie2/LacZ BM-transplanted diabetic versus nondiabetic mice. BM from diabetic and nondiabetic mice was transplanted into nondiabetic mice, revealing that reendothelialization was impaired in the recipients of diabetic BM. To examine the relative roles of denuded artery versus EPCs in diabetes, we injected diabetic and nondiabetic EPCs intravenously after arterial injury in diabetic and nondiabetic mice. Diabetic EPCs recruitment to the neoendothelium was significantly reduced, regardless of the diabetic status of the recipient mice. In vitro, diabetic EPCs exhibited decreased migration and adhesion activities. Vascular endothelial growth factor and endothelial NO synthase expressions were also significantly reduced in diabetic EPCs. Notably, thrombospondin-1 mRNA expression was significantly upregulated in diabetic EPCs, associating with the decreased EPC adhesion activity in vitro and in vivo. Reendothelialization is impaired by malfunctioning EPCs in diabetes. Diabetic EPCs have phenotypic differences involving thrombospondin-1 expression compared with nondiabetic EPCs, revealing potential novel mechanistic insights and therapeutic targets to improve reendothelialization and reduce restenosis in diabetes.


Subject(s)
Diabetes Mellitus/physiopathology , Endothelial Cells/physiology , Stem Cells/physiology , Thrombospondin 1/physiology , Animals , Bone Marrow Transplantation , Cell Adhesion , Cell Movement , Cells, Cultured , Cytokines/biosynthesis , Male , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase Type III/physiology , Vascular Endothelial Growth Factor A/physiology
12.
J Exp Med ; 203(1): 153-63, 2006 Jan 23.
Article in English | MEDLINE | ID: mdl-16401693

ABSTRACT

The cell surface receptor alpha4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of alpha4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of alpha4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively alpha4 integrin-expressing cells. In vivo, a single dose of anti-alpha4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti-alpha4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti-alpha4 integrin ex vivo or collected from alpha4 integrin-deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that alpha4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of alpha4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.


Subject(s)
Integrin alpha4/metabolism , Myocardial Ischemia/physiopathology , Neovascularization, Physiologic , Stem Cells/physiology , Animals , Bone Marrow , Cell Movement , Endothelial Cells , Integrin alpha4/genetics , Male , Mice , Mice, Knockout
13.
Regen Med ; 1(3): 337-45, 2006 May.
Article in English | MEDLINE | ID: mdl-17465787

ABSTRACT

Since the first experiments of cell transplantation into the heart were performed in the early 1990s, the identification of adult stem cells has triggered attempts to regenerate damaged heart tissue by cellular transplantation. Until recently, a multitude of adult stem or progenitor cells from various tissues have been proposed to meet this end. Bone marrow in particular has emerged as the most promising source for stem and progenitor cells because, besides being the organ of hematopoietic maintenance, it contains a complex assortment of stem and progenitor cells. A large body of provocative experimental evidence for vascular and myocardial regeneration by these cells has generated further enthusiasm for their use. However, many questions remain unanswered in this new field of research regarding the therapeutic potential and the mechanisms responsible for the observed therapeutic effects. In this review, the authors discuss the therapeutic capacity of currently available representative bone marrow-derived stem and progenitor cells for treating ischemic heart diseases.


Subject(s)
Bone Marrow Cells/cytology , Myocardial Ischemia/therapy , Stem Cell Transplantation , Stem Cells/physiology , Adult , Cell Differentiation , Forecasting , Humans , Models, Cardiovascular , Myocardial Infarction/pathology , Myocardium/cytology , Myocytes, Cardiac , Neovascularization, Physiologic , Regeneration , Stem Cells/classification , Stem Cells/cytology , Tissue Donors
14.
Circulation ; 112(1): 93-102, 2005 Jul 05.
Article in English | MEDLINE | ID: mdl-15983249

ABSTRACT

BACKGROUND: Peripheral neuropathy is a frequent and major complication of diabetes. METHODS AND RESULTS: Severe peripheral neuropathy developed in type II diabetic mice, characterized by significant slowing of motor and sensory nerve conduction velocities. Rosuvastatin restored nerve vascularity, including vessel size, and nerve function also recovered to the levels of nondiabetic mice. Neuronal nitric oxide synthase expression in sciatic nerves was reduced in diabetic mice but was preserved by rosuvastatin. Coadministration of a nitric oxide synthase inhibitor with rosuvastatin attenuated the beneficial effects of rosuvastatin on nerve function and limited the recovery of vasa nervorum and nerve function. In vitro, rosuvastatin inhibited downregulation of neuronal nitric oxide synthase expression induced by high-glucose conditions in cultured Schwann cells. Furthermore, Akt phosphorylation in Schwann cells, downregulated by high-glucose conditions, was also restored by rosuvastatin, consistent with the change of neuronal nitric oxide synthase expression. Akt inhibition independently reduced neuronal nitric oxide synthase expression in Schwann cells in low-glucose cultures. CONCLUSIONS: These data indicate that the HMG-CoA reductase inhibitor rosuvastatin has a favorable effect on diabetic neuropathy independent of its cholesterol-lowering effect. Our data provide evidence that this effect may be mediated in part via neuronal nitric oxide synthase/nitric oxide and phosphatidylinositol 3-kinase/Akt-signaling pathways and also suggest that restoration or preservation of the microcirculation of the sciatic nerve may be involved.


Subject(s)
Diabetic Nephropathies/drug therapy , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Nitric Oxide Synthase Type I/physiology , Vasa Nervorum/drug effects , Animals , Cells, Cultured , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/drug therapy , Down-Regulation/drug effects , Fluorobenzenes/administration & dosage , Fluorobenzenes/pharmacology , Glucose/pharmacology , Male , Mice , Mice, Mutant Strains , Nitric Oxide Synthase Type I/genetics , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/administration & dosage , Pyrimidines/pharmacology , Rosuvastatin Calcium , Schwann Cells/drug effects , Schwann Cells/metabolism , Sulfonamides/administration & dosage , Sulfonamides/pharmacology , Treatment Outcome
15.
Vasc Med ; 10(1): 15-22, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15920995

ABSTRACT

Hyperhomocyst(e)inemia (HH) is an established independent risk factor for coronary, cerebral and peripheral vascular diseases. Recent studies have indicated that certain cardiovascular risk factors, including diabetes and hypercholesterolemia, impair expression of vascular endothelial growth factor (VEGF) and endogenous angiogenesis. In this study, we investigate the impact of moderate HH on angiogenesis and VEGF pathway in a mouse model of hindlimb ischemia. Upon induction of unilateral hindlimb ischemia, endogenous angiogenesis, expression of VEGF, and phosphorylation of the VEGF receptor Flk-1 were evaluated in mice heterozygous for a deletion of the cystathionine beta-synthase gene (CBS) and compared with those observed in CBS+/+ mice. CBS+/- mice exhibit moderate HH, as demonstrated by measuring plasma total homocyst(e)ine (tHcy) levels, which were significantly higher in these animals compared with CBS+/+ mice (4.77 +/- 0.82 vs 2.10 +/- 0.28, p < 0.01). Twenty-eight days after induction of ischemia, hindlimb blood flow was significantly reduced in CBS+/- mice compared with CBS+/+ animals (0.49 +/- 0.03, n = 12 vs 0.71 +/- 0.09, n = 10; p < 0.05). In addition, there was a significant negative correlation between plasma homocyst(e)ine levels and the laser Doppler perfusion ratio in CBS+/- mice (p = 0.0087, r = -0.7171). While VEGF expression and Flk-1 phosphorylation were not impaired in the ischemic muscles of CBS+/- mice, phosphorylation of the endothelial cell survival factor Akt was significantly inhibited by homocyst(e)ine in a dose-dependent manner in human umbilical vein endothelial cell (HUVECs) in vitro. In conclusion, our findings demonstrate that endogenous angiogenesis is inversely related to plasma levels of homocyst(e)ine in genetically engineered, heterozygous mice with moderate HH. This impairment, however, is not dependent on reduced expression of VEGF or impaired phosphorylation of its receptor Flk-1. In contrast, our data suggest that impaired Akt phosphorylation mediates the impairment of angiogenesis associated with HH.


Subject(s)
Hyperhomocysteinemia/physiopathology , Ischemia/blood , Neovascularization, Physiologic/physiology , Animals , Disease Models, Animal , Mice , Mice, Inbred Strains , Phosphorylation , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism
16.
Circulation ; 111(20): 2662-70, 2005 May 24.
Article in English | MEDLINE | ID: mdl-15897348

ABSTRACT

BACKGROUND: Toxic neuropathies induced by cisplatin and other chemotherapeutic agents are important clinical problems because of their high incidence, their lack of effective treatment, and the fact that neuropathy represents a dose-limiting factor for these therapies. The pathogenic basis for toxic neuropathies induced by chemotherapeutic agents has not been completely elucidated. METHODS AND RESULTS: We investigated the hypothesis that experimental toxic neuropathy results from an antiangiogenic effect of these drugs, resulting in destruction of the vasa nervorum, and accordingly that the neuropathy could be prevented or reversed by locally administered VEGF gene transfer without augmenting tumor growth. In an animal model of cisplatin-induced neuropathy, nerve blood flow was markedly attenuated, and there was a profound reduction in the number of vasa nervorum associated with marked endothelial cell apoptosis, resulting in a severe peripheral neuropathy with focal axonal degeneration characteristic of ischemic neuropathy. After intramuscular gene transfer of naked plasmid DNA encoding VEGF-1 in animals with an established neuropathy, vascularity and blood flow returned to levels similar to those of control rats, peripheral nerve function was restored, and histological nerve architecture was normalized. Gene therapy administered in parallel with cisplatin chemotherapy completely attenuated endothelial cell apoptosis and inhibited destruction of nerve vasculature, deterioration of nerve function, and axonal degeneration. In a rat tumor model, VEGF gene transfer administered locally did not alter tumor growth or vascularity. CONCLUSIONS: These findings implicate microvascular damage as the basis for toxic neuropathy induced by cisplatin and suggest that local angiogenic gene therapy may constitute a novel prevention or treatment for this disorder without augmenting tumor growth or vascularization.


Subject(s)
Cisplatin/adverse effects , Peripheral Nervous System Diseases/prevention & control , Vascular Endothelial Growth Factor A/administration & dosage , Angiogenesis Inhibitors/adverse effects , Animals , Apoptosis , Endothelium, Vascular/cytology , Genetic Therapy , Humans , Male , Neoplasms/blood supply , Neoplasms/complications , Neoplasms/drug therapy , Peripheral Nervous System Diseases/chemically induced , Peripheral Nervous System Diseases/therapy , Rats , Rats, Sprague-Dawley , Tumor Burden/drug effects , Umbilical Veins/cytology , Vasa Nervorum/drug effects , Vascular Endothelial Growth Factor A/therapeutic use
17.
J Clin Invest ; 115(2): 326-38, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15690083

ABSTRACT

We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis.


Subject(s)
Bone Marrow Cells/physiology , Cell Differentiation/physiology , Heart/physiology , Multipotent Stem Cells/transplantation , Myocardial Infarction/therapy , Regeneration/physiology , Adult , Animals , Bone Marrow Cells/cytology , Cell Lineage/physiology , Cell Proliferation , Cells, Cultured , Female , Humans , Male , Multipotent Stem Cells/physiology , Myocardial Infarction/pathology , Myocardium/pathology , Myocytes, Cardiac/pathology , Myocytes, Cardiac/physiology , Myocytes, Smooth Muscle/pathology , Myocytes, Smooth Muscle/physiology , Neovascularization, Physiologic/physiology , Rats , Rats, Nude
18.
Circulation ; 111(9): 1114-20, 2005 Mar 08.
Article in English | MEDLINE | ID: mdl-15723985

ABSTRACT

BACKGROUND: The function of bone marrow-derived endothelial progenitor cells (EPCs) in repair of ischemic tissue has been the subject of intense scrutiny, and the capacity of these cells to contribute significantly to new blood vessels remains controversial. The possibility that EPCs could act as reservoirs of cytokines has been implied by several observations; however, a specific role for cytokine delivery has not been identified. METHODS AND RESULTS: We performed a series of experiments that revealed the rapid recruitment of EPCs to the myocardium by very short periods of ischemia, so-called ischemic preconditioning. The recruited EPCs express an array of potentially cardioprotective cytokines including nitric oxide synthase isoforms. Bone marrow transplantation studies, using donor marrow null for nitric oxide synthase isoforms, revealed that both endothelial and inducible nitric oxide synthase derived from bone marrow cells play essential roles in the cardioprotective effect that normally occurs after ischemic preconditioning. CONCLUSIONS: These findings provide novel insights about the role of bone marrow-derived cells in ischemic preconditioning and also reveal that distinct mechanisms regulate recovery after ischemia-reperfusion and chronic ischemic injury.


Subject(s)
Bone Marrow Transplantation , Endothelial Cells/enzymology , Ischemic Preconditioning, Myocardial , Myocardial Infarction/pathology , Myocardial Ischemia/physiopathology , Nitric Oxide Synthase/physiology , Nitric Oxide/physiology , Stem Cells/enzymology , Animals , Blood Cells/cytology , Blood Cells/enzymology , Bone Marrow Cells/cytology , Bone Marrow Cells/enzymology , Capillaries/pathology , Cell Hypoxia , Cell Movement , Cells, Cultured/enzymology , Coronary Circulation , Cytokines/physiology , Endothelial Cells/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/prevention & control , Myocardium/enzymology , Myocardium/pathology , Neovascularization, Physiologic , Nitric Oxide Synthase/deficiency , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Phosphorylation , Protein Processing, Post-Translational , Radiation Chimera , Stem Cells/cytology , Vascular Endothelial Growth Factor A/physiology
19.
Circulation ; 110(1): 36-45, 2004 Jul 06.
Article in English | MEDLINE | ID: mdl-15210598

ABSTRACT

BACKGROUND: Drug-eluting stents represent a useful strategy for the prevention of restenosis using various antiproliferative drugs. These strategies share the liability of impairing endothelial recovery, thereby altering the natural biology of the vessel wall and increasing the associated risk of stent thrombosis. Accordingly, we tested the hypothesis that local delivery via gene-eluting stent of naked plasmid DNA encoding for human vascular endothelial growth factor (VEGF)-2 could achieve similar reductions in neointima formation while accelerating, rather than inhibiting, reendothelialization. METHODS AND RESULTS: phVEGF 2-plasmid (100 or 200 microg per stent)-coated BiodivYsio phosphorylcholine polymer stents versus uncoated stents were deployed in a randomized, blinded fashion in iliac arteries of 40 normocholesterolemic and 16 hypercholesterolemic rabbits. Reendothelialization was nearly complete in the VEGF stent group after 10 days and was significantly greater than in control stents (98.7+/-1% versus 79.0+/-6%, P<0.01). At 3 months, intravascular ultrasound analysis revealed that lumen cross-sectional area (4.2+/-0.4 versus 2.27+/-0.3 mm(2), P<0.001) was significantly greater and percent cross-sectional narrowing was significantly lower (23.4+/-6 versus 51.2+/-10, P<0.001) in VEGF stents compared with control stents implanted in hypercholesterolemic rabbits. Transgene expression was detectable in the vessel wall along with improved functional recovery of stented segments, resulting in a 2.4-fold increase in NO production. CONCLUSIONS: Acceleration of reendothelialization via VEGF-2 gene-eluting stents provides an alternative treatment strategy for the prevention of restenosis. VEGF-2 gene-eluting stents may be considered as a stand-alone or combination therapy.


Subject(s)
Arterial Occlusive Diseases/prevention & control , Gene Transfer Techniques , Genetic Therapy/methods , Stents , Vascular Endothelial Growth Factors/genetics , Animals , Arterial Occlusive Diseases/diagnostic imaging , Arterial Occlusive Diseases/pathology , Combined Modality Therapy , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Gene Expression , Male , Nitric Oxide/biosynthesis , Plasmids/administration & dosage , Rabbits , Stem Cells/cytology , Ultrasonography , Vascular Endothelial Growth Factors/analysis
20.
J Clin Invest ; 111(5): 717-25, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12618526

ABSTRACT

Although lymphedema is a common clinical condition, treatment for this disabling condition remains limited and largely ineffective. Recently, it has been reported that overexpression of VEGF-C correlates with increased lymphatic vessel growth (lymphangiogenesis). However, the effect of VEGF-C-induced lymphangiogenesis on lymphedema has yet to be demonstrated. Here we investigated the impact of local transfer of naked plasmid DNA encoding human VEGF-C (phVEGF-C) on two animal models of lymphedema: one in the rabbit ear and the other in the mouse tail. In a rabbit model, following local phVEGF-C gene transfer, VEGFR-3 expression was significantly increased. This gene transfer led to a decrease in thickness and volume of lymphedema, improvement of lymphatic function demonstrated by serial lymphoscintigraphy, and finally, attenuation of the fibrofatty changes of the skin, the final consequences of lymphedema. The favorable effect of phVEGF-C on lymphedema was reconfirmed in a mouse tail model. Immunohistochemical analysis using lymphatic-specific markers: VEGFR-3, lymphatic endothelial hyaluronan receptor-1, together with the proliferation marker Ki-67 Ab revealed that phVEGF-C transfection potently induced new lymphatic vessel growth. This study, we believe for the first time, documents that gene transfer of phVEGF-C resolves lymphedema through direct augmentation of lymphangiogenesis. This novel therapeutic strategy may merit clinical investigation in patients with lymphedema.


Subject(s)
Endothelial Growth Factors/genetics , Genetic Therapy , Lymphatic System/physiology , Lymphedema/therapy , Neovascularization, Physiologic/drug effects , Amino Acid Sequence , Animals , Disease Models, Animal , Immunohistochemistry , Molecular Sequence Data , Phosphorylation , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , Transgenes , Vascular Endothelial Growth Factor C , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factor Receptor-3/genetics , Vascular Endothelial Growth Factor Receptor-3/metabolism
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