Role of VPAC1 and VPAC2 in VIP mediated inhibition of rat pulmonary artery and aortic smooth muscle cell proliferation.

Recent studies have suggested the potential use of vasoactive intestinal peptide (VIP) in the treatment of pulmonary arterial hypertension (PAH). An understanding of the mechanism of action of VIP is important for the development of new therapies for PAH. The biological effects of VIP are mediated by two type II guanine nucleotide binding protein (G-protein)-coupled receptors VIP/PACAP (pituitary adenylate cyclase activating peptide) receptor type1 (VPAC1) and VIP/PACAP receptor type 2 (VPAC2). In the present study, the distribution and role of these receptors were investigated and compared in cultured smooth muscle cells from rat aorta and pulmonary artery, as well as in fixed tissue sections of the aorta and pulmonary artery. Western blot analysis, RT-PCR and immunohistochemistry showed the expression of both VIP receptors in tissue sections of the aorta and pulmonary artery as well as in cultured smooth muscle cells from these vessels. The application of a specific antagonist of VPAC1 resulted in a small release from VIP induced inhibition of cell proliferation. In contrast (VIP 6-28; 300nM) which is an antagonist against both receptors resulted in a significant restoration of proliferation. The expression of cAMP was reduced in the presence of VIP 6-28 and slightly decreased by VPAC1 antagonist. These findings suggest a dual role for VPAC1 and VPAC2 receptors in mediating the antiproliferative effects of VIP with VPAC2 appearing to play a more dominant role.

Adenoviral transfer of vasoactive intestinal peptide (VIP) gene inhibits rat aortic and pulmonary artery smooth muscle cell proliferation.

Vasoactive intestinal peptide (VIP), a 28 amino acid peptide, has been shown to inhibit proliferation of vascular smooth muscle cells. In previous studies VIP and VIP analogs have been used to study the effects of the peptide on vascular smooth muscle cell function. In this study an adenovirus encoding the VIP gene was used to investigate the mechanism of the antiproliferative action of VIP in vascular smooth muscle cells. Primary cultures of aortic and pulmonary artery smooth muscle cells from male Sprague-Dawley rats were transfected with varying concentrations of serotype 5 adenovirus encoding human VIP (Ad5CMVhVIP). Transfection efficiency and subsequently VIP gene expression were confirmed by western blot analysis and immunohistochemistry. In this study a decrease in vascular smooth muscle cell proliferation at vector concentrations of 150, 300 and 600MOI (multiplicity of infection) was observed. In addition, there was increased production of cAMP in pulmonary artery and aortic smooth muscle cells transfected with VIP. Treatment of cells with a PKA inhibitor (Rp-8-BrcAMPs) restored proliferation to about 80% of control whereas treatment with the PKG inhibitor Rp-8-BrcGMPs had no significant effect suggesting the involvement of the PKA pathway in the antiproliferative actions of VIP.

Inhibition of vascular smooth muscle cell proliferation in vitro by genetically engineered marrow stromal cells secreting calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP) has a beneficial effect in pulmonary hypertension and is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs), also known as mesenchymal stem cells, hold promise for use in adult stem cell-based ex vivo gene therapy. To test the hypothesis that genetically engineered MSCs secreting CGRP can inhibit vascular smooth muscle cell proliferation, rat MSCs were isolated, ex vivo expanded, and transduced with adenovirus containing CGRP. Immunocytochemical analysis demonstrated that wild type rat MSCs express markers specific for stem cells, endothelial cells, and smooth muscle cells including Thy-1, c-Kit, von Willebrand Factor and alpha-smooth muscle actin. Immunocytochemistry confirmed the expression of CGRP by the transduced rat MSCs. The transduced rat MSCs released 10.3+/-1.3 pmol CGRP/1 x 10(6) cells/48 h (mean+/-S.E.M., n=3) into culture medium at MOI 300 and the CGRP-containing culture supernatant from the transduced cells inhibited the proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and rat aortic smooth muscle cells (ASMCs) in culture. Co-culture of the transduced rat MSCs with rat PASMCs or rat ASMCs also inhibited smooth muscle cell proliferation. These findings suggest that this novel adult stem cell-based CGRP gene therapy has potential for the treatment of cardiovascular diseases including pulmonary hypertension.

Rosiglitazone reduces serum homocysteine levels, smooth muscle proliferation, and intimal hyperplasia in Sprague-Dawley rats fed a high methionine diet.

Homocysteine (Hcy) is a metabolite of the essential amino acid methionine. Hyperhomocysteinemia is associated with vascular disease, particularly carotid stenosis. Rosiglitazone, a ligand of the peroxisome proliferator-activated receptor gamma , attenuates balloon catheter-induced carotid intimal hyperplasia in type 2 diabetic rats. We studied 4 groups (n = 7 per group) of adult female Sprague-Dawley rats fed (a) powdered laboratory chow (control), (b) control diet with rosiglitazone (3.0 mg/kg/d), (c) diet containing 1.0% l -methionine, and (d) diet containing methionine and rosiglitazone. After 1 week on high methionine diet, the rats were administered an aqueous preparation of rosiglitazone by oral gavage. One week after initiation of rosiglitazone, balloon catheter injury of the carotid artery was carried out using established methods, and the animals continued on their respective dietary and drug regimens for another 21 days. At the end of the experimental period, blood samples were collected, and carotid arteries and liver were harvested. Serum Hcy increased significantly on methionine diet compared with controls (28.9 +/- 3.2 vs 6.3 +/- 0.04 micromol/L). Development of intimal hyperplasia was 4-fold higher in methionine-fed rats; this augmentation was significantly reduced ( P < .018) in rosiglitazone-treated animals. Rosiglitazone treatment significantly ( P < .001) suppressed Hcy levels and increased the activity of the Hcy metabolizing enzyme, cystathionine-beta-synthase in the liver samples. Hcy (100 micromol/L) produced a 3-fold increase in proliferation of rat aortic vascular smooth muscle cells; this augmentation was inhibited by incorporating rosiglitazone (10 micromol/L). After balloon catheter injury to the carotid artery of animals on a high methionine diet, there was an increase in the rate of development of intimal hyperplasia consistent with the known effects of Hcy. It is demonstrated for the first time that the peroxisome proliferator-activated receptor gamma agonist rosiglitazone can attenuate the Hcy-stimulated increase in the rate of development of intimal hyperplasia indirectly by increasing the rate of catabolism of Hcy by cystathionine-beta-synthase and directly by inhibiting vascular smooth muscle cell proliferation. These findings may have important implications for the prevention of cardiovascular disease and events in patients with hyperhomocysteinemia (HHcy).

Engineering ex vivo-expanded marrow stromal cells to secrete calcitonin gene-related peptide using adenoviral vector.

Calcitonin gene-related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated CGRP gene transfer into ex vivo-expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro-CGRP and AdntlacZ, adenoviral vectors containing prepro-CGRP or nuclear-targeted beta-galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral-mediated gene transfer into ex vivo-expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro- CGRP-transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro-CGRP-transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 +/- 0.4 pmol CGRP/1 x 10(6) cells/48 hours (mean +/- standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro-CGRP-transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication-deficient recombinant adenovirus can be used to gene engineer ex vivo-expanded MSCs and that high-level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell-based cell and gene therapy strategy for the treatment of cardiovascular diseases.

Adenoviral gene transfer of eNOS: high-level expression in ex vivo expanded marrow stromal cells.

Endothelial nitric oxide synthase (eNOS) is an attractive target for cardiovascular gene therapy. Marrow stromal cells (MSCs), also known as mesenchymal stem cells, hold great promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated eNOS gene transfer into ex vivo expanded MSCs, rat MSCs (rMSCs) were isolated, expanded ex vivo, and transduced with Ad5RSVeNOS, an adenoviral vector containing the eNOS gene under the control of the Rous sarcoma virus promoter. The presence of eNOS protein in Ad5RSVeNOS-transduced rMSCs was confirmed by immunohistochemical and Western blot analysis. Transduction efficiency was dose dependent, and eNOS transgene expression in rMSCs persisted for > or =21 days in culture. The rMSCs retained multipotential differentiation capability after adenoviral-mediated eNOS gene transfer. Furthermore, intracavernosal injection of Ad5RSVeNOS-transduced rMSCs increased the expression of eNOS in the corpus cavernosum, and stem cells were identified within corporal sinusoids. These findings demonstrate that replication-deficient recombinant adenovirus can be used to engineer ex vivo expanded rMSCs and that high-level eNOS transgene expression can be achieved, pointing out the clinical potential of using this novel adult stem cell-based gene therapy method for the treatment of cardiovascular diseases.

Mechanism of eNOS gene transfer inhibition of vascular smooth muscle cell proliferation.

Endothelial nitric oxide synthase (eNOS) is responsible for the production of nitric oxide (NO) in blood vessels. NO has been shown to be involved in the inhibition of vascular smooth muscle cell (VSMC) proliferation. In the present study, the eNOS gene was transferred into rat aortic smooth muscle cells by using an adenoviral vector, and the effect of endogenously produced NO on VSMC proliferation was investigated. The presence of eNOS in eNOS-transfected cells was confirmed by immunocytochemistry and Western blot analysis. eNOS transfection resulted in inhibition of VSMC proliferation. This effect was accompanied by increased levels of p53 and p21. This effect was abrogated in the presence of the protein kinase A (PKA) inhibitor Rp-8-bromoadenosine 3',5'-cyclic monophosphothioate. The increased levels of p53 and p21 observed in eNOS-transfected cells were reduced in the presence of the PKA inhibitor. These data suggest that p21 and p53 play a role in the inhibition of proliferation in eNOS-transfected cells and that levels of these two proteins are regulated by PKA.