Muscle Cell Proliferation In Response Research Articles

Spatiotemporal ATF3 Expression Determines VSMC Fate in Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. ATF3 (activating transcription factor 3) has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 in AAA development and progression remains elusive. Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adeno-associated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor β) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.

Dual specificity phosphatase 6 promotes vascular smooth muscle cell proliferation in response to inflammatary stimulation and vascular injury

Dual specificity phosphatase 6 promotes vascular smooth muscle cell proliferation in response to inflammatary stimulation and vascular injury

Local Delivery of miR-21 Stabilizes Fibrous Caps in Vulnerable Atherosclerotic Lesions.

miRNAs are potential regulators of carotid artery stenosis andconcordant vulnerable atherosclerotic plaques. Hence, we analyzed miRNA expression in laser captured micro-dissected fibrous caps of either ruptured or stable plaques (n= 10 each), discovering that miR-21 was significantly downregulated in unstable lesions. To functionally evaluate miR-21 in plaque vulnerability, miR-21 and miR-21/apolipoprotein-E double-deficient mice (Apoe-/-miR-21-/-) were assessed. miR-21-/- mice lacked sufficient smooth muscle cell proliferation inresponse to carotid ligation injury. When exposing Apoe-/-miR-21-/- mice to an inducible plaque rupture model,they presented with more atherothrombotic events (93%) compared with miR-21+/+Apoe-/- mice (57%). We discovered that smooth muscle cell fate in experimentally induced advanced lesions is steered via a REST-miR-21-REST feedback signaling pathway. Furthermore, Apoe-/-miR-21-/- mice presented with more pronounced atherosclerotic lesions, greater foam cell formation, and substantially higher levels of arterial macrophage infiltration. Local delivery of a miR-21 mimic using ultrasound-targeted microbubbles into carotid plaques rescued the vulnerable plaque rupture phenotype. Inthe present study, we identify miR-21 as a key modulator of pathologic processes in advanced atherosclerosis. Targeted, lesion site-specific overexpression of miR-21 can stabilize vulnerable plaques.

Mechano growth factor, a splice variant of IGF-1, promotes neurogenesis in the aging mouse brain

Mechano growth factor (MGF) is a splice variant of IGF-1 first described in skeletal muscle. MGF induces muscle cell proliferation in response to muscle stress and injury. In control mice we found endogenous expression of MGF in neurogenic areas of the brain and these levels declined with age. To better understand the role of MGF in the brain, we used transgenic mice that constitutively overexpressed MGF from birth. MGF overexpression significantly increased the number of BrdU+ proliferative cells in the dentate gyrus (DG) of the hippocampus and subventricular zone (SVG). Although MGF overexpression increased the overall rate of adult hippocampal neurogenesis at the proliferation stage it did not alter the distribution of neurons at post-mitotic maturation stages. We then used the lac-operon system to conditionally overexpress MGF in the mouse brain beginning at 1, 3 and 12 months with histological and behavioral observation at 24 months of age. With conditional overexpression there was an increase of BrdU+ proliferating cells and BrdU+ differentiated mature neurons in the olfactory bulbs at 24 months when overexpression was induced from 1 and 3 months of age but not when started at 12 months. This was associated with preserved olfactory function. In vitro, MGF increased the size and number of neurospheres harvested from SVZ-derived neural stem cells (NSCs). These findings indicate that MGF overexpression increases the number of neural progenitor cells and promotes neurogenesis but does not alter the distribution of adult newborn neurons at post-mitotic stages. Maintaining youthful levels of MGF may be important in reversing age-related neuronal loss and brain dysfunction.

S2-3 BRG1-dependent epigenetic control of vascular smooth muscle cell proliferation by hydrogen sulfide

Hydrogen sulfide (H 2 S) can modulate the proliferation of vascular smooth muscle cells. This study was designed to investigate the epigenetic control of vascular smooth muscle cell proliferation in response to H 2 S. Microarray analysis indicated that brahma-related gene 1 ( Brg1 ) and proliferation-related genes including proliferating cell nuclear antigen ( Pcna ), neurotrophin 3 ( Ntf3 ) and platelet-derived growth factor subunit A ( Pdgfα ) were significantly downregulated by H 2 S in endothelin-1-stimulated proliferative vascular smooth muscle cells. Brg1 is the central catalytic subunit of the SWI/SNF apparatus (an ATP-dependent chromatin remodeling complex). Overexpression and knockdown of Brg1 confirmed that Brg1 was crucial for H 2 S-induced inhibition of vascular smooth muscle cell proliferation. A luciferase reporter assay, real-time PCR and Western blotting demonstrated that H 2 S inhibited Brg1 transcription and expression. A DNase I hypersensitivity assay revealed that H 2 S reversed endothelin-1-stimulated Pcna , Ntf3 and Pdgfα chromatin remodeling and vascular smooth muscle cell proliferation. A chromatin immunoprecipitation assay indicated that H 2 S inhibited the recruitment of Brg1 to the Pcna , Ntf3 and Pdgfα promoters. The results of this study indicate that H 2 S inhibits vascular smooth muscle cell proliferation via an epigenetic mechanism involving inhibition of Brg1 transcription and expression, and by reducing recruitment of Brg1 to the Pcna , Ntf3 and Pdgfα promoter regions.

Regulation of fibrillar collagen-mediated smooth muscle cell proliferation in response to chemical stimuli by telomere reverse transcriptase through c-Myc

Human telomerase reverse transcriptase (hTERT) and oncogene c-Myc have been shown to regulate cell proliferation. Our previous studies demonstrated that fibrillar collagen mediates vascular smooth muscle cell (SMC) cycle progression and proliferation in response to platelet-derived growth factor (PDGF)-BB and interleukin (IL)-1β. However, whether hTERT and c-Myc are involved in these fibrillar collagen-mediated SMC responses remain unclear. The present study elucidated the regulatory role of hTERT and c-Myc in PDGF-BB/IL-1β-induced cell cycle progression in SMCs on fibrillar collagen and its underlying mechanisms. Our results showed that PDGF-BB and IL-1β exert synergistic effects to induce hTERT expression, but not its activity, in human arterial SMCs on fibrillar collagen. This PDGF-BB/IL-1β-induced up-regulation of hTERT contributes to cell cycle progression in SMCs through the up-regulation of cyclin-dependent kinase-6 and down-regulations of p27KIP1 and p21CIP1. In addition, PDGF-BB/IL-1β induces up-regulation of c-Myc in SMCs on fibrillar collagen; this response is mediated by the increased binding of hTERT, which can form complexes with TPP1 and hnRNPK, to the guanine-rich region of the c-Myc promoter and consequently contributes to cell cycle progression in SMCs on fibrillar collagen. Moreover, the PDGF-BB/IL-1β-induced hTERT and c-Myc expressions are regulated by phosphatidylinositol 3-kinase/Akt in SMCs on fibrillar collagen. Our findings provide insights into the mechanisms by which hTERT and c-Myc regulates SMC cell cycle progression and proliferation on fibrillar collagen in response to chemical stimuli.

The cyclic AMP response element‐binding protein (CREB) mediates smooth muscle cell proliferation in response to angiotensin II

The cAMP response element-binding protein (CREB) is a transcription factor that mediates the cellular response to metabolic and mitogenic signals. Whether CREB contributes to vascular function has received little attention, especially in relation to the processes associated with atherosclerotic disease progression and restenosis. This study examined the involvement of CREB in the mitogenic actions of angiotensin II (AngII), a growth factor that promotes neointimal hyperplasia in response to vascular injury. Treatments were performed on quiescent vascular smooth muscle cells (VSMCs) obtained from a porcine explant model. Organ culture was performed on porcine hearts subjected to angioplasty ex vivo. Stimulation of VSMCs with AngII resulted in transient CREB phosphorylation. Proliferation of smooth muscle cells in response to AngII was reduced by 90% after infection with adenovirus expressing dominant-negative killer CREB (kCREB) mutant. Likewise, expression of kCREB prevented angioplasty-induced neointimal hyperplasia. AngII-induced CREB phosphorylation was independent of cAMP activation. Examination of putative CREB kinases revealed that MSK was responsible for phosphorylating CREB. In addition, inhibition of PKC revealed that this kinase operates upstream and activates MSK. These results indicate that activation of CREB via PKC and MSK is essential for SMC proliferation in response to AngII.

Brg1-dependent epigenetic control of vascular smooth muscle cell proliferation by hydrogen sulfide

Hydrogen sulfide (H2S) can modulate the proliferation of vascular smooth muscle cells. This study was designed to investigate the epigenetic control of vascular smooth muscle cell proliferation in response to H2S. Microarray analysis indicated that Brahma-related gene 1 (Brg1) and proliferation-related genes including proliferating cell nuclear antigen (Pcna), neurotrophin 3 (Ntf3) and platelet-derived growth factor subunit A (Pdgfα) were significantly downregulated by H2S in endothelin-1-stimulated proliferative vascular smooth muscle cells. Brg1 is the central catalytic subunit of the SWI/SNF apparatus (an ATP-dependent chromatin remodeling complex). Overexpression and knockdown of Brg1 confirmed that Brg1 was crucial for H2S-induced inhibition of vascular smooth muscle cell proliferation. A luciferase reporter assay, real-time PCR and Western blotting demonstrated that H2S inhibited Brg1 transcription and expression. A DNase I hypersensitivity assay revealed that H2S reversed endothelin-1-stimulated Pcna, Ntf3 and Pdgfα chromatin remodeling and vascular smooth muscle cell proliferation. A chromatin immunoprecipitation assay indicated that H2S inhibited the recruitment of Brg1 to the Pcna, Ntf3 and Pdgfα promoters. The results of this study indicate that H2S inhibits vascular smooth muscle cell proliferation via an epigenetic mechanism involving the inhibition of Brg1 transcription and expression, and by reducing the recruitment of Brg1 to the Pcna, Ntf3 and Pdgfα promoter regions.

Effect of cyclic hydrodynamic pressure‐induced proliferation of human bladder smooth muscle through Ras‐related C3 botulinum toxin substrate 1, mitogen‐activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2

To examine the role of Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 in the cyclic hydrodynamic pressure-induced proliferation of human bladder smooth muscle cells. Human bladder smooth muscle cells were exposed to cyclic hydrodynamic pressures in vitro with defined parameters (static, 100 cmH(2) O, 200 cmH(2) O and 300 cmH(2) O pressure) for 24 h. The proliferation of cells was assessed by flow cytometry. Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 messenger ribonucleic acid, and protein expression was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the Rac1 was determined with real-time polymerase chain reaction and Western blot technique with small interfering ribonucleic acid transfection and Rac1 inhibitor (NSC23766). The proliferation of human bladder smooth muscle cells was increased. Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 were activated by 200 and 300 cmH(2) O cyclic hydrodynamic pressure compared with static and 100 cmH(2) O pressure. The "knockdown" of activation of Rac1 using target small interfering ribonucleic acid transfection and Rac1 inhibitor (NSC23766) decreased proliferation of human bladder smooth muscle cells, and downregulated mitogen-activated protein kinase kinase 1/2, extracellular regulated protein kinases 1/2. The Rac1 pathway is activated in mechanotransduction and regulation of human bladder smooth muscle cell proliferation in response to cyclic hydrodynamic pressure.

Activation of the sonic hedgehog signaling controls human pulmonary arterial smooth muscle cell proliferation in response to hypoxia

The hedgehog signal pathway plays a crucial role in the angiogenesis and vascular remodeling. However, the function of this pathway in the pulmonary vascular smooth cell proliferation in response to hypoxia remains unknown. In this study, we have demonstrated that the main components of the hedgehog pathway, including sonic hedgehog (SHH), patched1 (PTCH1), smoothened (SMO), GLI and hypoxia-inducible factor 1 (HIF1) are expressed in the human pulmonary arterial smooth muscle cells (HPASMCs). Interestingly, hypoxia significantly enhanced the expression of SHH and HIF1, facilitated the translocation of GLI1 into the nuclei, and promoted the proliferation of HPASMCs. Furthermore, direct activation of the SHH pathway through incubation with the purified recombinant human SHH or with purmorphamine and SAG, two Smo agonists, also enhanced the proliferation of HPASMCs. Importantly, the treatment with anti-SHH and anti-HIF1 antibodies or cyclopamine, a specific SMO inhibitor, markedly inhibited the nuclear translocation of GLI1 and cell proliferation in the HPASMCs induced by hypoxia and activation of the SHH pathway. Moreover, the treatment with cyclopamine increased apoptosis in the hypoxic HPASMCs. These data strongly demonstrate for the first time that the SHH signaling plays a crucial role in the regulation of HPASMC growth in response to hypoxia.

The Physiological Significance of p27 KIP1 Expression in Detrusor Function

Bladder outlet obstruction results in smooth muscle cell hyperplasia, decreased bladder wall compliance, and lower and upper urinary tract pathology. The cyclin-dependent kinase inhibitor p27(KIP1) regulates bladder smooth muscle cell proliferation in response to bladder outlet obstruction but little is known about its physiological role in the bladder. We investigated the role of p27(KIP1) in the structure and function of the detrusor layer of the bladder wall. We used immunoblotting and reverse transcriptase-polymerase chain reaction to examine cell cycle regulation in response to increased mechanical tension in an in vitro model of tension induced smooth muscle cell proliferation and an in vivo model of bladder outlet obstruction. We compared unobstructed bladders of p27(+/+) and p27(-/-) mice (Jackson Laboratory, Bar Harbor, Maine) structurally by histological staining and functionally by in vivo cystometric measurements of bladder capacity, detrusor compliance and detrusor leak point pressure. Increased tension decreased p27(KIP1) at the protein level in human bladder smooth muscle cells and in intact murine bladder smooth muscle. p27(-/-) mice had bladder smooth muscle cell hyperplasia even in the absence of bladder outlet obstruction. While p27 loss had little effect on detrusor leak point pressure, p27(-/-) mice had significantly decreased bladder capacity and detrusor compliance. To our knowledge we provide the first report of the in vivo significance of p27(KIP1) in the regulation of detrusor function using a cystometric approach. We identified a role for p27(KIP1) in protecting against dysregulated smooth muscle cell proliferation, bladder capacity and detrusor compliance under normotensive conditions.

Differential Distribution of Podocan Isoforms

Podocan is a relatively novel member of the small leucine‐rich repeat family of proteins. First isolated from podocytes of HIV‐1‐infected kidney tissue, this protein seems to be involved in a number of biological pathways. Initial experiments in podocan‐knockout mice revealed increases in bone density and in vascular smooth muscle cell proliferation in response to injury when compared to the phenotypes of genetically unaltered mice. In addition, protein labeling of human kidney biopsies and vascular lesions has shown both intra and extracellular distribution of podocan. To determine if multiple isoforms of the protein exist, we began a bioinformatics study to align all reported isoforms of human podocan. We found that the previously reported 613 amino acid isoform had a strong N‐terminal signal peptide, as expected for a secreted protein. However, three other isoforms had additional amino acids in front of the signal peptide. We hypothesized that additional sequences upstream of the signal peptide would lead to retention of podocan in the cytoplasm. To test this hypothesis, we fused EGFP to the N‐ or C‐ terminus of secreted podocan. These constructs were subsequently transfected into renal 293T cells. We examined the cells by fluorescent microscopy and western blotting 48 hours later. As predicted, N‐terminal GFP‐podocan prevented efficient secretion of podocan. Our studies have led us to posit that podocan is involved in differential signaling, and this area will be the focus of future investigation. We have generated 3 C‐terminal His‐tagged podocan isoforms that have differing lengths of N‐terminal sequences upstream of the signal peptide and will use these to confirm our GFP‐fusion findings. We have also developed a series of isoform‐specific primers to help us determine whether these isoforms are differentially expressed in normal or diseased human tissue.Grant Funding SourceAmerican Association of Anatomists

Protein kinase CK1αLS mediates hydrogen peroxide stimulated vascular cell proliferation and intimal hyperplasia

Protein kinase CK1αLS and its substrate hnRNP‐C constitute a novel nuclear signaling pathway in vascular cells that is activated by mitogenic levels of hydrogen peroxide and is upregulated in intimal hyperplasia and atherosclerosis. The role of CK1αLS in vascular cell proliferation was assessed using retrovirally‐delivered shRNA to lower the expression of the kinase. Knock down of CK1αLS inhibited proliferation of human umbilical vein endothelial cells, human coronary artery endothelial cells, and human coronary artery smooth muscle cells (CASMC). Knock down of CK1αLS also abolished hydrogen peroxide stimulated proliferation of CASMC with accumulation of these cells in cell cycle phase G0/G1 by flow cytometry. Human internal thoracic arteries in culture developed smooth muscle cell proliferation, increased proteoglycan deposition, and intimal hyperplasia. All of these pro‐atherosclerotic changes were inhibited by knocking down CK1αLS. These findings indicate that protein kinase CK1αLS positively regulates vascular cell proliferation and intimal hyperplasia and specifically mediates vascular smooth muscle cell proliferation in response to low physiologic levels of hydrogen peroxide.

Phosphorylation of beta‐catenin by PKA promotes vascular smooth muscle cell proliferation in response to purinergic receptor stimulation.

Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of P2Y purinergic receptors. We have previously shown that a) ATP stimulates a transient activation of protein kinase A (PKA), which is required for maximal DNA synthesis in VSMC; and b) PKA can phosphorylate beta‐catenin at two novel sites (Ser552 and Ser675) in COS7‐overexpression cell model. Beta‐catenin is a signaling molecule which is regulated through degradation / stabilization mechanism, and which promotes cell proliferation by activation of T cell factor (TCF) transcription factors. In the present study, using the new phospho‐specific antibodies against pSer552 or pSer675 sites of beta‐catenin, we show that ATP can stimulate PKA‐dependent phosphorylation of endogenous beta‐catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA‐dependent stimulation of TCF transcriptional activity, in part, through an increased association of phosphorylated (by PKA) beta‐catenin with TCF‐4. Using adenovirus‐mediated transduction of PKA inhibitor, PKI, or of dominant negative TCF‐4 mutant, we show that ATP stimulates cyclin D1 promoter activation, cyclin D1 protein expression and proliferation of VSMC – all in PKA‐ and TCF‐dependent manner. In conclusion, we show a critical role of ATP‐induced phosphorylation of beta‐catenin by PKA in proliferation of VSMC.

Pitavastatin Suppresses Acute and Chronic Rejection in Murine Cardiac Allografts

HMG-CoA reductase inhibitors play several roles in the maintenance of organ transplants. We investigated the role of pitavastatin, a potent and newly developed HMG-CoA reductase inhibitor, in cardiac allograft rejection and mechanism of graft arterial disease (GAD) suppression. Balb/c mice hearts were transplanted into C3H/He mice (a full allomismatch combination) to assess acute rejection or C57BL/6 hearts into B6.C-H2(<bm12>)KhEg (a class II mismatch combination) to examine the extent of GAD. Pitavastatin was administered orally to mice everyday (3 mg/kg/day). To assess the effect in acute rejection, mixed lymphocyte reaction was performed and cytokine mRNA expression was examined with ribonuclease protection assay. Pitavastatin significantly prolonged allograft survival. Lymphocyte proliferation was inhibited by pitavastatin, and RPA showed down-regulation of interleukin-6 in pitavastatin-treated cardiac allografts. Allografts in the pitavastatin-treated group after 8 weeks showed less GAD compared with the control group. In vitro, pitavastatin suppressed the smooth muscle cell proliferation in response to activated T cells and inhibited extracellular signal-regulated kinase 1/2 activation. Pitavastatin could be effective in the suppression of acute rejection and GAD development in cardiac transplantation.

Insulin-Like Growth Factor-I Signaling in Smooth Muscle Cells Is Regulated by Ligand Binding to the 177CYDMKTTC184 Sequence of the β3-Subunit of αVβ3

The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.

Egr-1 in vascular smooth muscle cell proliferation in response to allo-antigen

Background Early growth response factor-1 (Egr-1) plays an important role in regulating multiple factors involved in the progression of vascular lesions. This study examined our hypothesis that Egr-1 plays a critical role in the early stage of chronic cardiac allograft rejection and in the proliferation of the smooth muscle cell response to alloantigen. Materials and methods Antisense Egr-1 oligodeoxynucleotide (ODN) was ex vivo gene transfected into the donor hearts from DBA/2 mice, followed by heterotopic allografting into B10.D2 recipients. The allografts were harvested on day 30. Egr-1 and its target molecules, such as platelet-derived growth factor (PDGF)-A, basic fibroblastic growth factor (bFGF), vascular cell adhesion molecule (VCAM)-1, transforming growth factor (TGF)-β and nonmuscle myosin heavy chain B (SMemb), were identified immunohistochemically, and the percentage of the lumen occluded by the intima was calculated. For the cell proliferation assay, sensitized T cells were harvested from B10.D2 recipients as stimulator and then added to the SMCs, which were harvested from DBA/2 mouse aorta. Cellular proliferation was measured and Egr-1 and its target gene expression were examined by real-time RT-PCR. Results Egr-1 and its target genes were expressed in the thickened intima from untreated recipients. Egr-1 antisense ODN inhibited not only Egr-1 expression but also its target genes and significantly suppressed intimal thickening of coronary arteries. Egr-1 antisense ODN also significantly inhibited cell proliferation and expressions of Egr-1 and Egr-1 target genes in a mixed cell culture model. Conclusion We conclude that Egr-1 plays an important role in the formation of the cardiac allograft vasculopathy responding to alloantigens.

CysLT1 receptor upregulation by TGF-β and IL-13 is associated with bronchial smooth muscle cell proliferation in response to LTD4

Background: Airway remodeling is a feature of chronic asthma. It involves a number of structural changes, including bronchial smooth muscle cell (BSMC) hyperplasia and hypertrophy. Cysteinyl leukotrienes (cysLTs) have been suggested to play a role in airway remodeling in addition to their numerous other physiopathologic effects. Objectives: This work was aimed at characterizing the potential modulation of CysLT1 receptor expression by cytokines and the eventual functional relevance of this modulation. Methods: Expression of CysLT1 receptor was measured by flow cytometry and immunofluorescence microscopy. Transcripts were measured by RT-PCR and BSMC proliferation by crystal violet staining. Results: When human BSMC were exposed to transforming growth factor (TGF)-β, IL-13, or IFN-γ, their expression of CysLT1 receptor was significantly augmented in a time- and concentration-dependent manner. Interestingly, IL-4 had no significant effect on CysLT1 receptor expression in BSMC. Moreover, IL-13 and IFN-γ but not TGF-β were able to increase CysLT1 mRNA levels. Finally, when BSMC were pretreated with TGF-β or IL-13 but not IFN-γ, their responsiveness to LTD4 was markedly enhanced in terms of BSMC proliferation. Whereas TGF-β, IL-13, or LTD4 alone had little effect on BSMC proliferation, preexposure of the cells to TGF-β or IL-13 for 24 hours resulted in a significant increase in proliferation in response to LTD4. The enhanced proliferation was totally prevented by pretreating the cytokine-primed BSMC with the selective CysLT1 receptor antagonist Montelukast. Conclusions: Taken together, our findings indicate a synergy between certain cytokines and cysLTs, mediated by the augmented expression of the CysLT1 receptor and subsequent LTD4-triggered BSMC proliferation. These findings support a role for cysLTs in the airway remodeling observed in asthmatic patients and may provide a rationale for preventive and therapeutic intervention.

CysLT1 receptor upregulation by TGF-beta and IL-13, but not IL-4, is associated with bronchial smooth muscle cell proliferation in response to LTD4

CysLT1 receptor upregulation by TGF-beta and IL-13, but not IL-4, is associated with bronchial smooth muscle cell proliferation in response to LTD4

G-protein expression and intimal hyperplasia after arterial injury: A role for Gαi proteins

Purpose: Guanine nucleotide binding protein (G-protein) coupled receptors are involved in smooth muscle cell proliferation, but the role of G-proteins in arterial intimal hyperplasia has not been defined. This study examines the expression of G-proteins in the developing intimal hyperplasia after balloon injury of the rat carotid artery and specifically tests the hypothesis that the pertussis toxin sensitive Gi G-protein subunit plays a role in the initiation of intimal hyperplasia. Methods: In vitro responses to serum stimulation (10% fetal bovine serum) were examined in the presence and absence of pertussis toxin (PTx). After a standard balloon injury in male Sprague-Dawley rats, the expression of G-protein subunits (αo, αi, αq, αs, and βγ) was determined by means of Western blotting in the first 28 days. Thereafter, a second set of animals was allocated to control and PTx-treated (a Gαi inhibitor; 500 ng/mL in an externally applied 30% pluronic gel) groups. Smooth muscle cell proliferation was estimated by means of thymidine analogue 5-bromo-2′ deoxyuridine incorporation 2 days after injury, and vessel dimensions were determined by means of videomorphometry 14 days after injury. Results: There was inhibition of DNA synthesis and smooth muscle cell proliferation in response to serum with an IC50 of 100 ng/mL. Three days after balloon injury, there was an increase in Gαi3 expression, which decreased at days 7, 14, and 28, compared with the uninjured carotid. Gαq expression increased in a time-dependent manner. There was a marked time-dependent increase in Gβγ in the 28 days. Gαi2 and Gαs isoforms (45 and 52 kDa) did not change significantly with time. There was no major change in Gαi1 and Gαo in the study period. At 14 days, PTx treatment reduced intimal hyperplasia by 52% (63 ± 4 μm vs 30 ± 5 μm, control vs PTx; P <.001). Medial smooth muscle cell proliferation at day 2 was decreased in the PTx group, compared with that in the gel-coated group (15% ± 2% and 26% ± 3%; P =.02). Conclusion: After balloon injury, there is a time-dependent increase in G-protein expression, which is subunit specific. Activation of PTx sensitive G-proteins (Gαi) is involved during the initiation of intimal hyperplasia after arterial injury, and their inhibition results in a decrease in early medial cell proliferation. This acute interruption of Gi signaling produces a long-term decrease in intimal hyperplasia. (J Vasc Surg 2001;33:408-18.)