Restricted expression of homeobox genes distinguishes fetal from adult human smooth muscle cells.

Smooth muscle cell plasticity is considered a prerequisite for atherosclerosis and restenosis following angioplasty and bypass surgery. Identification of transcription factors that specify one smooth muscle cell phenotype over another therefore may be of major importance in understanding the molecular basis of these vascular disorders. Homeobox genes exemplify one class of transcription factors that could govern smooth muscle cell phenotypic diversity. Accordingly, we screened adult and fetal human smooth muscle cell cDNA libraries with a degenerate oligonucleotide corresponding to a highly conserved region of the homeodomain with the idea that homeobox genes, if present, would display a smooth muscle cell phenotype-dependent pattern of expression. No homeobox genes were detected in the adult human smooth muscle cell library; however, five nonparalogous homeobox genes were uncovered from the fetal library (HoxA5, HoxA11, HoxB1, HoxB7, and HoxC9). Northern blotting of adult and fetal tissues revealed low and restricted expression of all five homeobox genes. No significant differences in transcripts of HoxA5, HoxA11, and HoxB1 were detected between adult or fetal human smooth muscle cells in culture. HoxB7 and HoxC9, however, showed preferential mRNA expression in fetal human smooth muscle cells that appeared to correlate with the age of the donor. This phenotype-dependent expression of homeobox genes was also noted in rat pup versus adult smooth muscle cells. While similar differences in gene expression have been reported between subsets of smooth muscle cells from rat vessels of different-aged animals or clones of rat smooth muscle, our findings represent a demonstration of a transcription factor distinguishing two human smooth muscle cell phenotypes.

Oxygen-sensitive calcium channels in vascular smooth muscle and their possible role in hypoxic arterial relaxation.

We have investigated the modifications of cytosolic [Ca2+] and the activity of Ca2+ channels in freshly dispersed arterial myocytes to test whether lowering O2 tension (PO2) directly influences Ca2+ homeostasis in these cells. Unclamped cells loaded with fura-2 AM exhibit oscillations of cytosolic Ca2+ whose frequency depends on extracellular Ca2+ influx. Switching from a PO2 of 150 to 20 mmHg leads to a reversible attenuation of the Ca2+ oscillations. In voltage-clamped cells, hypoxia reversibly reduces the influx of Ca2+ through voltage-dependent channels, which can account for the inhibition of the Ca2+ oscillations. Low PO2 selectively inhibits L-type Ca2+ channel activity, whereas the current mediated by T-type channels is unaltered by hypoxia. The effect of low PO2 on the L-type channels is markedly voltage dependent, being more apparent with moderate depolarizations. These findings demonstrate the existence of O2-sensitive, voltage-dependent, Ca2+ channels in vascular smooth muscle that may critically contribute to the local regulation of circulation.

The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism.

Smooth muscle cell (SMC) proliferation is thought to play a major role in vascular restenosis after angioplasty and is a serious complication of the procedure. Developing antisense (AS) oligonucleotides as therapeutics is attractive because of the potentially high specificity of binding to their targets, and several investigators have reported inhibition of SMC proliferation in vitro and in vivo by using AS strategies. We report here the results of our experiments on vascular SMCs using AS oligonucleotides directed toward c-myb and c-myc. We found that significant inhibition of SMC proliferation occurred with these specific AS sequences but that this inhibition was clearly not via a hybridization-dependent AS mechanism. Rather, inhibition was due to the presence of four contiguous guanosine residues in the oligonucleotide sequence. This was demonstrated in vitro in primary cultures of SMCs and in arteries ex vivo. The ex vivo model developed here provides a rapid and effective system in which to screen potential oligonucleotide drugs for restenosis. We have further explored the sequence requirements of this non-AS effect and determined that phosphorothioate oligonucleotides containing at least two sets of three or four consecutive guanosine residues inhibit SMC proliferation in vitro and ex vivo. These results suggest that previous AS data obtained using these and similar, contiguous guanosine-containing AS sequences be reevaluated and that there may be an additional class of nucleic acid compounds that have potential as antirestenosis therapeutics.

Investigating the Role of PDE4D1/2 in Vascular Smooth Muscle Cell Desensitization

Vascular smooth muscle cell (VSMC) behaviour and phenotypic modulation is critical to vessel repair following damage, and the progression of various cardiovascular diseases. The second messenger cyclic adenosine monophosphosphate (cAMP) plays a key role in VSMC function under the synthetic/activated phenotype, which is typically associated with unhealthy cell behaviour. Consequently, cAMP signaling is often targeted in attempts to impact several pathological diseases, including atherosclerosis, restenosis, and pulmonary arterial hypertension (PAH). The cyclic nucleotide phosphodiesterases (PDEs) catalyze hydrolysis of cAMP to an inactive form, and therefore directly regulate cAMP signaling. The PDE4D family dominates in synthetic VSMCs, and there is considerable interest in determining how distinct PDE4D isoforms affect cell function. Specifically, we are interested in the potential link between short isoforms of PDE4D and VSMC desensitization to pharmacological agents that impact cardiovascular disease via cAMP signaling. This study extends on previous work that assessed the expression of PDE4D splice variants in rat aortic VSMCs following prolonged challenge with cAMP-elevating agents. It was determined that PDE4D1 and PDE4D2 were uniquely expressed in synthetic VSMCs incubated with these agents, and that this upregulation impacted PDE activity and cAMP accumulation in these cells. Here, we report that PDE4D1 and PDE4D2 are markedly upregulated in synthetic human aortic smooth muscle cells (HASMCs) following prolonged challenge with cAMP-elevating agents. Using a combination of RNAi-based and pharmacological approaches, we establish that this upregulation is reflected in levels of cAMP PDE activity, and restricted to the cytosolic sub-cellular compartment. Our results suggest a role for localized PDE4D1 and PDE4D2 activity in regulating cAMP-mediated desensitization in HASMCs, and highlight their therapeutic potential in treating various cardiovascular diseases.

Differential mechanisms of angiotensin II and PDGF-BB on migration and proliferation of coronary artery smooth muscle cells

Angiotensin II (Ang II) and platelet-derived growth factor-BB (PDGF-BB) are associated with excessive cell migration, proliferation and many growth-related diseases. However, whether these agents utilise similar mechanisms to trigger vascular pathologies remains to be explored. The effects of Ang II and PDGF-BB on coronary artery smooth muscle cell (CASMC) migration and proliferation were investigated via Dunn chemotaxis assay and the measurement of [3H]thymidine incorporation rates, respectively. Both atherogens produced similar degrees of cell migration which were dramatically inhibited by mevastatin (10 nM). However, the inhibitory effects of losartan (10 nM) and MnTBAP (a free radical scavenger; 50 μM) were found to be unique to Ang II-mediated chemotaxis. In contrast, MnTBAP, apocynin (an antioxidant and phagocytic NADPH oxidase inhibitor; 500 μM), mevastatin and pravastatin (100 nM) equally suppressed both Ang II and PDGF-BB-induced cellular growth. Although atherogens produced similar changes in NADPH oxidase, NOS and superoxide dismutase activities, they differentially regulated antioxidant glutathione peroxidase activity which was diminished by Ang II and unaffected by PDGF-BB. Studies with signal transduction pathway inhibitors revealed the involvement of multiple pathways i.e. protein kinase C, tyrosine kinase and MAPK in Ang II- and/or PDGF-BB-induced aforementioned enzyme activity changes. In conclusion, Ang II and PDGF-BB may induce coronary atherosclerotic disease formation by stimulating CASMC migration and proliferation through agent-specific regulation of oxidative status and utilisation of different signal transduction pathways.

Investigating the Role of PDE4D1/2 in Vascular Smooth Muscle Cell Desensitization

Thesis (Master, Biochemistry) -- Queen's University, 2016-10-14 02:44:01.604

GABAB receptors are expressed in human aortic smooth muscle cells and regulate the intracellular Ca2+ concentration

The aim of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter gamma-aminobutyric acid (GABAB), in human aortic smooth muscle cells (HASMCs), and to explore if altering receptor activation modified intracellular Ca(2+) concentration ([Ca(2+)]i) of HASMCs. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABABR1 and GABABR2 in cultured HASMCs. Immunohistochemistry was used to localize the two subunits in human left anterior descending artery (LAD). The effects of the GABAB receptor agonist baclofen on [Ca(2+)]i in cultured HASMCs were demonstrated using fluo-3. Both GABABR1 and GABABR2 mRNA and protein were identified in cultured HASMCs and antibody staining was also localized to smooth muscle cells of human LAD. 100 μM baclofen caused a transient increase of [Ca(2+)]i in cultured HASMCs regardless of whether Ca(2+) was added to the medium, and the effects were inhibited by pre-treatment with CGP46381 (selective GABAB receptor antagonist), pertussis toxin (a Gi/o protein inhibitor), and U73122 (a phospholipase C blocker). GABAB receptors are expressed in HASMCs and regulate the [Ca(2+)]i via a Gi/o-coupled receptor pathway and a phospholipase C activation pathway

Isolation, characterization, and functional assessment of novel smooth muscle like stem progenitors

Vascular smooth muscle cells (VSMC) are one of the key players in the pathogenesis of cardiovascular diseases. The origin of neointimal VSMC has thus become a prime focus of research. VSMC originate from multiple progenitors cell types. In embryo the well-defined sources of VSMC include; neural crest cells, proepicardial cells and EPC. In adults, though progenitor cells from bone marrow (BM), circulation and tissues giving rise to SMC have been identified, no progress has been made in terms of isolating highly proliferative clonal population of adult stem cells with potential to differentiate into SMC. Smooth muscle like stem progenitor cells (SMSPC) were isolated from cardiopulmonary bypass filters of adult patients undergoing CABG. Rat SMSPC have previously been isolated by our group from the bone marrow of Fischer rats and also from the peripheral blood of monocrotaline induced pulmonary hypertension (MCT-PHTN) animal model. Characterization of novel SMSPC exhibited stem cell characteristics and machinery for differentiation into SMC. The expression of Isl-1 on SMSPC provided unique molecular identity to these circulating stem progenitor cells. The functional potential of SMSPC was determined by monitoring adoptive transfer of GFP+ SMSPC in rodent models of vascular injury; carotid injury and MCT-PHTN. The participation of SMSPC in vascular pathology was confirmed by quantifying the peripheral blood, and engrafted levels of SMSPC using RT-PCR. In terms of translating into clinical practice, SMSPC could be a good tool for detecting the atherosclerotic plaque burden. The current study demonstrates the existence of novel adult stem progenitor cells in circulation, with the potential role in vascular pathology.