Proliferation Of Aortic Smooth Muscle Cells Research Articles

Oral administration of the thrombin receptor antagonist E5555 (atopaxar) attenuates intimal thickening following balloon injury in rats

Thrombin is a powerful agonist for a variety of cellular responses including platelet aggregation and vascular smooth muscle cell (SMC) proliferation. These actions are mediated by a thrombin receptor known as protease-activated receptor-1 (PAR-1). Recently we discovered that 1-(3- tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2 H-isoindol-2-yl)ethanone hydrobromide (E5555, atopaxar) is a potent and selective thrombin receptor antagonist. This study characterized the pharmacological effects of E5555 on SMC proliferation in vitro and in a rat model of intimal thickening after balloon injury in vivo. E5555 selectively inhibited rat aortic SMC proliferation induced by thrombin and thrombin receptor-activating peptide (TRAP) with half maximal inhibitory concentration (IC 50) values of 0.16 and 0.038 μM, respectively. E5555 did not inhibit rat SMC proliferation induced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at concentrations up to 1 μM. In addition, E5555 inhibited human aortic SMC proliferation induced by thrombin at concentrations of 0.3 and 3 units/ml with IC 50 values of 0.028 and 0.079 μM, respectively, whereas it did not affect bFGF-induced proliferation at concentrations up to 1 μM. Repeated oral administration of 30 mg/kg E5555 (once daily for 16 days) significantly reduced neointimal formation in the balloon-injured rat arterial model. These results suggested that a PAR-1 antagonist could be effective for treating restenosis following vascular intervention in addition to preventing thrombus formation. E5555 could thus have therapeutic potential for restenosis and chronic atherothrombotic disease.

Involvement of Nitric Oxide in the Inhibition of Aortic Smooth Muscle Cell Proliferation by Calcium Dobesilate.

Vascular smooth muscle cell (SMC) proliferation is a key process in the pathogenesis of atherosclerosis. Numerous factors are involved in the regulation of SMC growth. Nitric oxide (NO) induces the inhibition of SMC proliferation whereas oxidized low-density lipoproteins (LDL) have a mitogenic effect. Calcium dobesilate (Doxium) is an angioprotective agent for treating vascular diseases. It has been shown to increase NO production and to have antioxidant properties but its mechanism of action is not yet fully understood. This study investigated the effect of calcium dobesilate on proliferation of rat aortic SMC in culture. Proliferation was evaluated by cell number and DNA synthesis. Orally administered calcium dobesilate (30, 100, or 200 mg/kg/day for 7 days) induced a dose-dependent decrease of proliferation of SMC in primary culture compared with controls. In vitro treatment with calcium dobesilate (0.05-5 mM) inhibited both DNA synthesis and proliferation in a time- and concentration-dependent manner. In both ex vivo and in vitro models, the inhibition was reversible upon removal of the drug. Calcium dobesilate also stimulated NO production and NO synthase activity. Inhibitors of NO synthesis attenuated the inhibitory effect of calcium dobesilate (300 µM) on DNA synthesis. In addition, calcium dobesilate (2.5-40 µM) induced a dose-dependent protection of cooper-induced LDL oxidation. These results showed that calcium dobesilate inhibits SMC proliferation, partly by a NO-dependent mechanism, and suggest that it could be effective in the treatment of pathological disorders associated with vascular SMC proliferation.

A new iridoid and effect on the rat aortic vascular smooth muscle cell proliferation of isolated compounds from Buddleja officinalis

A new iridoid, named methylscutelloside (1) together with 19 known compounds belonging to the iridoids (2-4), monoterpenoids (5), flavonoids (6-8), triterpenoids (9-14), and phenylethanoids (15-20) were isolated from the flowers of Buddleja officinalis. Their chemical structures were elucidated on the basis of physicochemical properties, and by spectroscopic methods including 1D, 2D NMR, and MS. All isolated compounds were tested in vitro for their effects on the proliferation of rat aortic vascular smooth muscle cells (VSMCs). Among them, iridoids were the main active components and showed significant inhibitory effects on PDGF-BB-induced proliferation in rat aortic VSMCs.

The FGF-2-Derived Peptide FREG Inhibits Melanoma Growth In Vitro and In Vivo

Previous data report that fibroblast growth factor-2 (FGF-2)-derived peptide FREG potently inhibits FGF-2-dependent angiogenesis in vitro and in vivo. Here, we show that FREG inhibits up to 70% in vitro growth and invasion/migration of smooth muscle and melanoma cells. Such inhibition is mediated by platelet-derived growth factor-receptor-α (PDGF-Rα); in fact, proliferation and migration were restored upon PDGF-Rα neutralization. Further experiments demonstrated that FREG interacts with PDGF-Rα both in vitro and in vivo and stimulates its phosphorylation. We have previously shown that overexpressing PDGF-Rα strongly inhibits melanoma growth in vivo; we, therefore, hypothesized that PDGF-Rα agonists may represent a novel tool to inhibit melanoma growth in vivo. To support this hypothesis, FREG was inoculated intravenously (i.v.) in a mouse melanoma model and markedly inhibited pulmonary metastases formation. Immunohistochemical analyses showed less proliferation, less angiogenesis, and more apoptosis in metastasized lungs upon FREG treatment, as compared to untreated controls. Finally, in preliminary acute toxicity studies, FREG showed no toxicity signs in healthy animals, and neither microscopic nor macroscopic toxicity at the liver, kidney, and lungs level. Altogether, these data indicate that FREG systemic treatment strongly inhibits melanoma metastases development and indicate for the first time that agonists of PDGF-Rα may control melanoma both in vitro and in vivo.

Potent Inhibition of Platelet-Derived Growth Factor-Stimulated Rat Aortic Vascular Smooth Muscle Cell Cycle and Proliferation by (2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-1-one, a Newly Synthesized Benzylideneacetophenone Derivative

One of the principal regulators of mitogenesis in vascular smooth muscle cells (VSMCs) is platelet-derived growth factor-BB (PDGF-BB). An increase of PDGF-BB expression has been observed in atherosclerotic lesions. The aim of this study was to elucidate the effects and molecular mechanism of (2E)-3-(4-hydroxy-3-methoxyphenyl) phenylpro-2-en-1-one (KTJ2242), a newly synthesized benzylideneacetophenone derivative, on PDGF-BB-stimulated rat aortic VSMCs. KTJ2242 induced accumulation of cells in the G1 phase of the cell cycle of VSMCs. We observed that KTJ2242 inhibited PDGF-BB-stimulated [3H]-thymidine incorporation into the DNA of VSMCs, and the cell number was significantly reduced in a concentration-dependent manner. Also, we observed that KTJ2242 decreased PDGF-BB-stimulated extracellular-regulated kinase 1 and 2 (ERK1/2) and Akt phosphorylation. These results suggest the possibility that KTJ2242 may be a potential agent with which to control vascular disorders and its antiproliferative mechanism may be mediated through partial Akt and ERK1/2-dependent signaling pathways.

Sensitive Fern (Onoclea sensibilis) Extract Suppresses Proliferation and Migration of Vascular Smooth Muscle Cells Inflamed by Neighboring Macrophages

Vascular smooth muscle cell (SMC) migration and proliferation contribute to arterial wound repair and thickening of the intimal layer in atherosclerosis. SMC can physically interact with monocytes and macrophages within the intima. This study evaluated whether macrophages modulated proliferation and migration of SMC in close proximity, which was suppressed by 1-25 µg/ml sensitive fern (Onoclea sensibilis) extract (SFE) inhibiting protein-tyrosine phosphatase-1B activity. The addition of conditioned media of THP-1-derived macrophages substantially promoted human aortic smooth muscle cell (HAoSMC) proliferation by ≈30%. HAoSMC proliferation was significantly attenuated by ≥10 µg/ml SFE most likely due to its diminution of platelet derived growth factor (PDGF)-BB secreted by neighbor macrophages. HAoSMC migration was also enhanced by culturing in THP-1 macrophage conditioned media, as evidenced by a scratch wound assay. However, the presence of ≥10 µg/ml SFE did not allow such migaration. When SFE was treated to THP-1 macrophages, the secretion of the adipokines, visfatin and resistin, was abrogated. SFE at 1-25 µg/ml dose-dependently diminished resistin-stimulated secretion of collagen IV and connective tissue growth factor (CTGF) in HAoSMC, indicating that macrophage resistin plays a role in the extracellular matrix (ECM) production of vascular SMC. These results demonstrate that SFE disturbed proliferation and migration of SMC instigated by inflammatory macrophages in close proximity. Therefore, this study provides novel information that SFE has the potential capability to prevent atherosclerosis involving SMC proliferation, migration and fibrogenic activation within the vessels.

Early hypercholesterolemia contributes to vasomotor dysfunction and injury associated atherogenesis that can be inhibited by nitric oxide

Atherosclerosis results in vasomotor dysfunction, in part, through impairment of nitric oxide (NO) dependent vasodilation. It is unclear whether blood vessels are dysfunctional in an early environment of hypercholesterolemia alone and if this contributes to the vascular injury response. We hypothesize that early hypercholesterolemia, prior to gross vascular changes, contributes to vasomotor dysfunction and the vascular injury response. The efficacy of NO therapy to protect against the injury response in this setting was also assessed. The effect of oxidized low density lipoprotein (oxLDL) and inducible NO synthase (iNOS) gene transfer on rat aortic smooth muscle cell (SMC) proliferation was measured with (3)H-thymidine incorporation. Common carotid arteries (CCA) from wild-type C57BL6 (WT or C57) and apolipoprotein E deficient (ApoE KO) mice fed normal or Western diets for 6 to 8 weeks were tested for vasomotor function using an arteriograph system. Studies were repeated after CCA injury. The effect of iNOS gene transfer on morphometry by histology and vasomotor responses in injured CCAs in ApoE KO was examined. OxLDL increased SMC proliferation by >50%. In SMC expressing iNOS, NO production was unaffected by oxLDL and reduced oxLDL and still inhibited SMC proliferation. Endothelium dependent vasorelaxation was reduced in uninjured CCAs from ApoE KO and C57 mice on the Western vs normal diet (ApoE 39% ± 2% vs 55% ± 13%; C57 50% ± 13% vs 76% ± 5%, P < .001) and was increased with longer durations of hypercholesterolemia. Endothelium-dependent and independent vasodilator responses were severely disrupted in C57 and ApoE KO mice 2 weeks following CCA injury but both recovered by 4 weeks. CCA injury in ApoE KO mice resulted in the formation of atheromatous lesions while C57 mice showed no change (intima 27,795 ± 1829 vs 237 ± 28 μm(2); media 46,306 ± 2448 vs 11,714 ± 392 μm(2), respectively; P < .001). This structural change in the ApoE KO reduced distensibility and increased stiffness. Finally, iNOS gene transfer to injured CCA in ApoE KO mice dramatically reduced atheromatous neointimal lesion formation. Early hypercholesterolemia impairs endothelial function, with severity being related to duration and magnitude of hypercholesterolemia. Severe hypercholesterolemia leads to atheromatous lesion formation following injury and stresses the role of vascular injury in atherogenesis and suggests different mechanisms are involved in endothelial dysfunction and the injury response. Despite these changes, iNOS gene transfer still effectively inhibits atheroma formation. These findings support early correction of hypercholesterolemia and emphasize the potential role for NO based therapies in disease states.

Downregulation of FHL1 Expression in Thoracic Aortic Dissection: Implications in Aortic Wall Remodeling and Pathogenesis of Thoracic Aortic Dissection

To investigate the potential role of four and a half Lin-11, Isl-1, and Mec-3 (FHL1) protein in the pathogenesis of thoracic aortic dissection (TAD). The expression levels and localization of FHL1 protein in aortic tissue of TAD were analyzed using Western blot and immunohistochemistry. Furthermore, small interfering ribonucleic acid was used to knock down the FHL1 gene in rat aortic smooth muscle cells (SMCs). After assessing knockdown efficiency and specificity by real-time polymerase chain reaction and Westernblot, the effect of FHL1 knockdown on cell proliferation and apoptosis was evaluated by 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay and flow cytometry, respectively. Compared with normal aortic tissue, FHL1 protein expression in aortic tissue from TAD patients was significantly downregulated. Immunohistochemistry analysis showed that FHL1 was mainly localized in the cytoplasm of SMCs. In diseased aortic tissue, FHL1 immunoreactivity was lowest in SMCs in the split aortic media and adjacent area, but relatively high in SMCs in the aortic intima and adventitia. FHL1 knockdown significantly inhibited the proliferation of rat aortic SMCs but exerted no obvious effect on cell apoptosis. FHL1 protein expression is downregulated in TAD. Downregulation of FHL1 expression might contribute to the pathogenesis of TAD, perhaps by suppressing the proliferation of aortic SMCs and affecting aortic wall remodeling.

A potential inhibitor of rat aortic vascular smooth muscle cell proliferation from the pollen of Typha angustata

By various chromatographic methods, three flavonoids, (2S)-naringenin (1), isorhamnetin 3-O-(2-O-α-L-rhamnopyranosyl) β-D-glucopyranoside (2), typhaneoside (3), and two sterol glycosides, β-sitosterol-3-O-(6-octadecanoyl) β-D-glucopyranoside (4) and β-sitosterol-3-O-(6-octadeca-9Z,12Z-dienoyl) β-D-glucopyranoside (5), were isolated from the pollen of Typha angustata. Their structures were determined on the basis of spectroscopic analyses. The flavonoids (1-3) were evaluated for their effects on the viability and proliferation of rat aortic smooth muscle cells. (2S)-naringenin (1) significantly inhibited cell proliferation in a dose-dependent manner without cytotoxic at concentrations of 30, and 50 μM; it reduced the number of cells following PDGF-BB treatment to 1.83 ± 0.30 × 10(4) and 2.20 ± 0.60 × 10(4) cells/well, respectively. These findings suggest that (2S)-naringenin has antiproliferative effects on aortic smooth muscle cells.

Betulinic acid inhibits high glucose‐induced vascular smooth muscle cells proliferation and migration

The proliferation of vascular smooth muscle cells may perform a crucial role in the pathogenesis of diabetic vascular disease. The principal objective of this study was to determine the effects of betulinic acid (BA) on human aortic smooth muscle cell (HASMC) proliferation induced by high glucose (HG). In this study, [(3) H]-thymidine incorporation under 25 mM HG was accelerated significantly as compared with 5.5 mM glucose, and this increase was inhibited significantly by BA treatment. We utilized Western blotting analysis to evaluate the effects of BA on cell-cycle regulatory proteins. HG induced the expression of cyclins/CDKs and reduced the expression of p21(waf1/cip1) /p27(kip1). However, BA also attenuated the expression of HG-induced cell-cycle regulatory proteins. The results of gelatin zymography demonstrated that the HG-treated HASMC secreted gelatinases, probably including MMP-2/-9, which may be involved in the invasion and migration of HASMC. Additionally, BA suppressed the protein and mRNA expression levels of MMP-2/-9 in a dose-dependent manner. BA inhibited the production of HG-induced hydrogen peroxide (H(2)O(2)) and the formation of DCF-sensitive intracellular reactive oxygen species (ROS). Further, BA suppressed the nuclear translocation and phosphorylation of IκB-α of NF-κB under HG conditions. Our results showed that BA exerts multiple effects on HG-induced HASMC proliferation and migration, including the inhibition of both MMP-2 and MMP-9 transcription, protein activity, and the downregulation of ROS/NF-κB signaling, thereby suggesting that BA may be a possible therapeutic approach to the inhibition of diabetic vascular disease.

Cigarette Smoke Extracts Promote Vascular Smooth Muscle Cell Proliferation and Enhances Contractile Responses in the Vasculature and Airway

Cigarette smoke exposure is a strong risk factor for cardiovascular and respiratory diseases. However, the knowledge about how cigarette smoke induces damage to vasculature and airway is limited. The present study was designed to examine the effects of cigarette smoke particles extracted by heptane (heptane-soluble smoke particles, HSP), by water (water-soluble smoke particles, WSP) and by DMSO (DMSO-soluble smoke particles, DSP), which represent lipophilic, hydrophilic and ambiphoteric constituents from the cigarette smoke, respectively. Human aortic smooth muscle cell (HASMC) proliferation was assessed in cell culture. Rat resistance artery and airway contractile responses to serotonin, U46619, phenylephrine, noradrenaline, acetylcholine, des-Arg⁹-bradykinin, bradykinin, sarafotoxin 6c and endothelin-1 were monitored by a sensitive myograph system. Immunocytochemistry and cell-based phosphoELISA assay were used to demonstrate activation of extracellular signal-regulated kinases 1/2 (ERK1/2). For the first time, our results demonstrate that although all the three extracts promote HASMC proliferation, the HSP and DSP effects occur earlier. HSP and DSP, but not WSP, increase the contractile responses to sarafotoxin 6c, U46619 or bradykinin in rat mesenteric artery and/or in bronchi. ERK1/2 is activated by HSP and DSP in HASMCs and inhibition of ERK1/2 abrogated the smoke extracts-induced HASMC proliferation, while blockage of nicotinic receptors had no effects, suggesting that the toxic effects of the smoke extracts occur via activation of intracellular ERK1/2 signalling, but not nicotinic receptors.

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; 300 nM) 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.

Chromosome 9p21 and Coronary Artery Disease

Genomewide association studies have implicated a region on chromosome 9p21 in coronary artery disease. This association is probably driven by two genes that mediate the proliferation of aortic smooth-muscle cells.

Angiotensin II–Induced Vascular Smooth Muscle Cell Migration and Growth Are Mediated by Cytochrome P450 1B1–Dependent Superoxide Generation

Cytochrome P450 1B1, expressed in vascular smooth muscle cells, can metabolize arachidonic acid in vitro into several products including 12- and 20-hydroxyeicosatetraenoic acids that stimulate vascular smooth muscle cell growth. This study was conducted to determine whether cytochrome P450 1B1 contributes to angiotensin II-induced rat aortic smooth muscle cell migration, proliferation, and protein synthesis. Angiotensin II stimulated migration of these cells, measured by the wound healing approach, by 1.78-fold; and DNA synthesis, measured by [(3)H]thymidine incorporation, by 1.44-fold after 24 hours; and protein synthesis, measured by [(3)H]leucine incorporation, by 1.40-fold after 48 hours. Treatment of vascular smooth muscle cells with the cytochrome P450 1B1 inhibitor 2,4,3',5'-tetramethoxystilbene or transduction of these cells with adenovirus cytochrome P450 1B1 small hairpin RNA but not its scrambled control reduced the activity of this enzyme and abolished angiotensin II- and arachidonic acid-induced cell migration, as well as [(3)H]thymidine and [(3)H]leucine incorporation. Metabolism of arachidonic acid to 5-, 12-, 15-, and 20-hydoxyeicosatetraenoic acids in these cells was not altered, but angiotensin II- and arachidonic acid-induced reactive oxygen species production and extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activity were inhibited by 2,4,3',5'-tetramethoxystilbene and cytochrome P450 1B1 small hairpin RNA (shRNA) and by Tempol, which inactivates reactive oxygen species. Tempol did not alter cytochrome P450 1B1 activity. These data suggest that angiotensin II-induced vascular smooth muscle cell migration and growth are mediated by reactive oxygen species generated from arachidonic acid by cytochrome P450 1B1 and activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase.

Pterostilbene, a natural dimethylated analog of resveratrol, inhibits rat aortic vascular smooth muscle cell proliferation by blocking Akt-dependent pathway

Vascular smooth muscle cells (VSMCs) are the main cellular component in the arterial wall, and abnormal proliferation of VSMCs plays a central role in the pathogenesis of atherosclerosis and restenosis after angioplasty, and possibly in the development of hypertension. Pterostilbene, a natural dimethylated analog of resveratrol, is known to have diverse pharmacological activities including anti-cancer, anti-inflammation and anti-oxidant activities. The present study was designed to investigate the effects of pterostilbene on platelet-derived growth factor (PDGF)-BB-induced VSMCs proliferation as well as the molecular mechanisms of the antiproliferative effects. The cell growth of VSMCs was determined by cell counting and [ 3H]thymidine incorporation assays. Pterostilbene significantly inhibited the DNA synthesis and proliferation of PDGF-BB-stimulated VSMCs in a concentration-dependent manner. The inhibition percentages of pterostilbene at 1, 3 and 5 μM to VSMCs proliferation were 68.5, 80.7 and 94.6%, respectively. The DNA synthesis of pterostilbene at 1, 3 and 5 μM in VSMCs was inhibited by 47.4, 76.7 and 100%, respectively. Pterostilbene inhibited the PDGF-BB-stimulated phosphorylation of Akt kinase. However, pterostilbene did not change the expression of extracellular signal-related kinase (ERK) 1/2, PLCγ1, phosphatidylinositol (PI)3 kinase and PDGF-Rβ phosphorylation. In addition, pterostilbene down-regulated the cell cycle-related proteins including the expression of cyclin-dependent kinase (CDK) 2, cyclin E, CDK4, cyclin D1, retinoblastoma (Rb) proteins and proliferative cell nuclear antigen (PCNA). These findings suggest that the inhibition of pterostilbene to the cell proliferation and DNA synthesis of PDGF-BB-stimulated VSMCs may be mediated by the suppression of Akt kinase. Furthermore, pterostilbene may be a potential anti-proliferative agent for the treatment of atherosclerosis and angioplasty restenosis.

Molecular mechanism of green microalgae,Dunaliella salina, involved in attenuating balloon injury-induced neointimal formation

The pathological mechanism of restenosis is primarily attributed to excessive proliferation of vascular smooth muscle cells (VSMC). The preventive effects of ethanol extract of Dunaliella salina (EDS) on balloon injury-induced neointimal formation were investigated. To explore its molecular mechanism in regulating cell proliferation, we first showed that EDS markedly reduced the human aortic smooth muscle cell proliferation via the inhibition of 5'-bromo-2'-deoxyuridine (BrdU) incorporation at 40 and 80 microg/ml. This was further supported by the G0/G1-phase arrest using a flow cytometric analysis. In an in vivo study, EDS at 40 and 80 microg/ml was previously administered to the Sprague-Dawley rats and found that the thickness of neointima, and the ratio of neointima:media were also reduced. EDS inhibited VSMC proliferation in a dose-dependent manner following stimulation of VSMC cultures with 15 % fetal bovine serum (FBS). Suppressed by EDS were 15 % FBS-stimulated intracellular Raf, phosphorylated extracellular signal-regulated kinases (p-Erk) involved in cell-cycle arrest and proliferating cell nuclear antigen. Phosphorylated focal adhesion kinase (p-FAK) was also suppressed by EDS. Also active caspase-9, caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels were increased by administration with EDS; the apoptotic pathway may play an important role in the regulatory effects of EDS on cell growth. These observations provide a mechanism of EDS in attenuating cell proliferation, thus as a potential intervention for restenosis.

Effect of betulinic acid on high glucose‐induced vascular smooth muscle cells proliferation

Betulinic acid (BA) is a pentacyclic triterpene found in Lycopus lucidus Turcz. Hyperglycemia has been shown to stimulate vascular smooth muscle cells proliferation, a paramount feature in atherosclerosis. The aim of this study was to determine the effects of BA on high glucose‐induced human aortic smooth muscle cell (HASMC) proliferation. In this study, [3H]‐thymidine incorporation under 25 mM glucose (HG) was significantly accelerated compared with 5.5 mM glucose and this increase was inhibited by BA. Invasion and migration assay also suggested an anti‐proliferative effect of BA. BA elevated the protein content of p21waf1/cip1, p27kip1, decreased cyclin D1/E and CDK2/4, suggesting that BA inhibited HASMC proliferation by regulating the cell progression from G1 to S phase. We showed that BA significantly inhibited HG‐induced matrix metalloproteinase (MMP)‐2/‐9 activity in a dose‐dependent manner. BA inhibited the HG‐induced hydrogen peroxide production and DCF‐sensitive intracellular ROS formation. Furthermore, BA suppressed the translocation and promoter transcriptional activity of NF‐κB under HG condition. BA also decreased HG‐induced phosphorylation of IκB‐α. In conclusion, the present data suggested that BA regulated cell cycle regulatory proteins, and significantly reduced HASMC proliferation, via inhibition of ROS and NF‐κB activation in hyperglycemia conditions.

Expression and characterization of a Grifola frondosa hydrophobin in Pichia pastoris

Hydrophobins are small secreted proteins produced by filamentous fungi. Being amphipathic and self-assembling, hydrophobins have drawn great attention since their discovery. The increase of production can reduce the cost and open up several new applications of hydrophobins. We successfully expressed recombinant Class I hydrophobin HGFI (rHGFI) by using pPIC9 vector with an alcohol oxidase 1 promoter in Pichia pastoris. Tricine-SDS–PAGE and Western blotting demonstrated that rHGFI, an 8kDa protein, was secreted into the culture medium. The culture conditions of the transformant strain were optimized by controlling the methanol concentration and induction time. Ultrafiltration and reverse-phase high performance liquid chromatography were used to perform a large-scale purification of rHGFI. A stable production of rHGFI around 86mg/L was achieved after the two-step purification. X-ray photoelectron spectroscopy and water contact angle measurements indicated that the functional rHGFI could self-assemble on hydrophobic siliconized glass and Teflon as well as on hydrophilic mica surfaces. A methylthiazol tetrazolium assay showed that rHGFI film could facilitate human aortic smooth muscle cell proliferation due to its cytocompatibility.

Spatiotemporal controlled delivery of nanoparticles to injured vasculature

There are a number of challenges associated with designing nanoparticles for medical applications. We define two challenges here: (i) conventional targeting against up-regulated cell surface antigens is limited by heterogeneity in expression, and (ii) previous studies suggest that the optimal size of nanoparticles designed for systemic delivery is approximately 50-150 nm, yet this size range confers a high surface area-to-volume ratio, which results in fast diffusive drug release. Here, we achieve spatial control by biopanning a phage library to discover materials that target abundant vascular antigens exposed in disease. Next, we achieve temporal control by designing 60-nm hybrid nanoparticles with a lipid shell interface surrounding a polymer core, which is loaded with slow-eluting conjugates of paclitaxel for controlled ester hydrolysis and drug release over approximately 12 days. The nanoparticles inhibited human aortic smooth muscle cell proliferation in vitro and showed greater in vivo vascular retention during percutaneous angioplasty over nontargeted controls. This nanoparticle technology may potentially be used toward the treatment of injured vasculature, a clinical problem of primary importance.

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

IntroductionVasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts.MethodsThe transgenic mouse strain Tβ RIIΔk-fib is characterized by balanced ligand-dependent upregulation of TGF-β signaling. Aortic and cardiac tissues were examined with histologic, biochemical, and isolated organ bath studies. Vascular and perivascular architecture was examined by hematoxylin and eosin (H&E) and special stains including immunostaining for TGF-β1 and phospho-Smad2/3 (pSmad2/3). Confirmatory aortic smooth muscle cell proliferation, phenotype, and functional assays, including signaling responses to exogenous TGF-β and endothelin-1, were performed. Aortic ring contractile responses to direct and receptor-mediated stimulation were assessed.ResultsAortic ring contractility and relaxation were diminished compared with wild-type controls, and this was associated with aortic adventitial fibrosis confirmed histologically and with Sircol assay. TGF-β1 and pSmad 2/3 expression was increased in the adventitia and smooth muscle layer of the aorta. Aortic smooth muscle cells from transgenic animals showed significant upregulation of TGF-β- responsive genes important for cytoskeletal function, such as transgelin and smoothelin, which were then resistant to further stimulation with exogenous TGF-β1. These cells promoted significantly more contraction of free floating type I collagen lattices when compared with the wild-type, but were again resistant to exogenous TGF-β1 stimulation. Aortic ring responses to receptor-mediated contraction were reduced in the transgenic animals. Specifically, bosentan reduced endothelin-mediated contraction in wild-type animals, but had no effect in transgenic animals, and endothelin axis gene expression was altered in transgenic animals. Transgenic mice developed cardiac fibrosis.ConclusionsThe histologic, biochemical, and functional phenotype of this transgenic mouse model of scleroderma offers insight into the altered biomechanical properties previously reported for large elastic arteries in human SSc and suggests a role for perturbed TGF-β and endothelin activity in this process.