ABSTRACT
The effect of intratracheal administration of cyclooxygenase-1 (COX-1)-modified adipose stem cells (ASCs) on monocrotaline-induced pulmonary hypertension (MCT-PH) was investigated in the rat. The COX-1 gene was cloned from rat intestinal cells, fused with a hemagglutanin (HA) tag, and cloned into a lentiviral vector. The COX-1 lentiviral vector was shown to enhance COX-1 protein expression and inhibit proliferation of vascular smooth muscle cells without increasing apoptosis. Human ASCs transfected with the COX-1 lentiviral vector (ASCCOX-1) display enhanced COX-1 activity while exhibiting similar differentiation potential compared with untransduced (native) ASCs. PH was induced in rats with MCT, and the rats were subsequently treated with intratracheal injection of ASCCOX-1 or untransduced ASCs. The intratracheal administration of ASCCOX-1 3 × 10(6) cells on day 14 after MCT treatment significantly attenuated MCT-induced PH when hemodynamic values were measured on day 35 after MCT treatment whereas administration of untransduced ASCs had no significant effect. These results indicate that intratracheally administered ASCCOX-1 persisted for at least 21 days in the lung and attenuate MCT-induced PH and right ventricular hypertrophy. In addition, vasodilator responses to the nitric oxide donor sodium nitroprusside were not altered by the presence of ASCCOX-1 in the lung. These data emphasize the effectiveness of ASCCOX-1 in the treatment of experimentally induced PH.
Subject(s)
Adipose Tissue/cytology , Adult Stem Cells/metabolism , Cyclooxygenase 1/metabolism , Hypertension, Pulmonary/therapy , Stem Cell Transplantation , Adult Stem Cells/cytology , Adult Stem Cells/transplantation , Animals , Cell Differentiation , Cyclooxygenase 1/genetics , Genetic Vectors/genetics , Humans , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/metabolism , Lentivirus/genetics , Monocrotaline/toxicity , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Aorta/metabolism , Cell Proliferation/drug effects , Myocytes, Smooth Muscle/physiology , Pulmonary Artery/metabolism , Receptors, Vasoactive Intestinal Peptide, Type II/physiology , Receptors, Vasoactive Intestinal Polypeptide, Type I/physiology , Vasoactive Intestinal Peptide/physiology , Animals , Aorta/cytology , Blotting, Western , Cells, Cultured , Cyclic AMP/metabolism , Gene Expression , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/physiopathology , Immunohistochemistry , Male , Peptide Fragments/pharmacology , Pulmonary Artery/cytology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Vasoactive Intestinal Peptide, Type II/antagonists & inhibitors , Receptors, Vasoactive Intestinal Peptide, Type II/genetics , Receptors, Vasoactive Intestinal Polypeptide, Type I/antagonists & inhibitors , Receptors, Vasoactive Intestinal Polypeptide, Type I/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Adenoviridae/genetics , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Vasoactive Intestinal Peptide/physiology , 8-Bromo Cyclic Adenosine Monophosphate/analogs & derivatives , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Blotting, Western , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Genetic Vectors , Hypertension, Pulmonary/drug therapy , Immunohistochemistry , Male , Rats , Rats, Sprague-Dawley , Thionucleotides/pharmacology , Vasoactive Intestinal Peptide/geneticsABSTRACT
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.
