Aspirin alleviates fibronectin-induced preeclampsia phenotypes in a mouse model and reverses fibronectin-mediated trophoblast invasiveness under hypoxia by regulating ciliogenesis and Akt and MAPK signaling.

The placenta experiences a low-oxygen stage during early pregnancy. Aspirin is an effective preventative treatment for preeclampsia if applied early in pregnancy. Elevation of fibronectin (FN) level has been reported to be associated with preeclampsia; however, the role of FN in the physiological hypoxic phase and whether aspirin exerts its effect on FN at this hypoxic stage remain unknown. We determined pregnancy outcomes by injecting saline or recombinant FN protein into C57BL/6 pregnant mice and one group of FN-injected mice was fed aspirin. The effects of FN, the underlying pathways on trophoblast biology, and cilia formation under hypoxia were investigated in FN-pretreated or FN-knockdown HTR-8/SVneo cells in a hypoxic chamber (0.1 % O2). Preeclampsia-like phenotypes, including blood pressure elevation and proteinuria, developed in FN-injected pregnant mice. The fetal weight of FN-injected mice was significantly lower than that of non-FN-injected mice (p < 0.005). Trophoblast FN expression was upregulated under hypoxia, which could be suppressed by aspirin treatment. FN inhibited trophoblast invasion and migration under hypoxia, and this inhibitory effect occurred through downregulating ZEB1/2, MMP 9 and the Akt and MAPK signaling pathways. Ciliogenesis of trophoblasts was stimulated under hypoxia but was inhibited by FN treatment. Aspirin was shown to reverse the FN-mediated inhibitory effect on trophoblast invasion/migration and ciliogenesis. In conclusion, FN overexpression induces preeclampsia-like symptoms and impairs fetal growth in mice. Aspirin may exert its suppressive effect on FN upregulation and FN-mediated cell function in the hypoxic stage of pregnancy and therefore provides a preventative effect on preeclampsia development.

3D-Printed proangiogenic patches of photo-crosslinked gelatin and polyurethane hydrogels laden with vascular cells for treating vascular ischemic diseases.

Engineering vascularized tissues remains a promising approach for treating ischemic cardiovascular diseases. The availability of 3D-bioprinted vascular grafts that induce therapeutic angiogenesis can help avoid necrosis and excision of ischemic tissues. Here, using a combination of living cells and biodegradable hydrogels, we fabricated 3D-printed biocompatible proangiogenic patches from endothelial cell-laden photo-crosslinked gelatin (EC-PCG) bioink and smooth muscle cell-encapsulated polyurethane (SMC-PU) bioink. Implantation of 3D-bioprinted proangiogenic patches in a mouse model showed that EC-PCG served as an angiogenic capillary bed, whereas patterned SMC-PU increased the density of microvessels. Moreover, the assembled patterns between EC-PCG and SMC-PU induced the geometrically guided generation of microvessels with blood perfusion. In a rodent model of hindlimb ischemia, the vascular patches rescued blood flow to distal tissues, prevented toe/foot necrosis, promoted muscle remodeling, and increased the capillary density, thereby improving the heat-escape behavior of ischemic animals. Thus, our 3D-printed vascular cell-laden bioinks constitute efficient and scalable biomaterials that facilitate the engineering of vascular patches capable of directing therapeutic angiogenesis for treating ischemic vascular diseases.

Endothelial Yin Yang 1 Phosphorylation at S118 Induces Atherosclerosis Under Flow.

RATIONALE: Disturbed flow occurring in arterial branches and curvatures induces vascular endothelial cell (EC) dysfunction and atherosclerosis. We postulated that disturbed flow plays important role in modulating phosphoprotein expression profiles to regulate endothelial functions and atherogenesis. OBJECTIVE: The goal of this study is to discover novel site-specific phosphorylation alterations induced by disturbed flow in ECs to contribute to atherosclerosis. METHODS AND RESULTS: Quantitative phosphoproteomics analysis of ECs exposed to disturbed flow with low and oscillatory shear stress (0.5±4 dynes/cm2) versus pulsatile shear stress (12±4 dynes/cm2) revealed that oscillatory shear stress induces phospho-YY1S118 (serine [S]118 phosphorylation of Yin Yang 1) in ECs. Elevated phospho-YY1S118 level in ECs was further confirmed to be present in the disturbed flow regions in experimental animals and human atherosclerotic arteries. This disturbed flow-induced EC phospho-YY1S118 is mediated by CK2α (casein kinase 2α) through its direct interaction with YY1. Yeast 2-hybrid library screening and in situ proximity ligation assays demonstrate that phospho-YY1S118 directly binds ZKSCAN4 (zinc finger with KRAB [krüppel-associated box] and SCAN [SRE-ZBP, CTfin51, AW-1 and Number 18 cDNA] domains 4) to induce promoter activity and gene expression of HDM2 (human double minute 2), which consequently induces EC proliferation through downregulation of p53 and p21CIP1. Administration of apoE-deficient (ApoE-/-) mice with CK2-specific inhibitor tetrabromocinnamic acid or atorvastatin inhibits atherosclerosis formation through downregulations of EC phospho-YY1S118 and HDM2. Generation of novel transgenic mice bearing EC-specific overexpression of S118-nonphosphorylatable mutant of YY1 in ApoE-/- mice confirms the critical role of phospho-YY1S118 in promoting atherosclerosis through EC HDM2. CONCLUSIONS: Our findings provide new insights into the mechanisms by which disturbed flow induces endothelial phospho-YY1S118 to promote atherosclerosis, thus indicating phospho-YY1S118 as a potential molecular target for atherosclerosis treatment.

Low Levels of MicroRNA-10a in Cardiovascular Endothelium and Blood Serum Are Related to Human Atherosclerotic Disease.

BACKGROUND: MicroRNA-10a (miR-10a) inhibits transcriptional factor GATA6 to repress inflammatory GATA6/VCAM-1 signaling, which is regulated by blood flow to affect endothelial function/dysfunction. This study aimed to identify the expression patterns of miR-10a/GATA6/VCAM-1 in vivo and study their implications in the pathophysiology of human coronary artery disease (CAD), i.e., atherosclerosis. METHODS: Human atherosclerotic coronary arteries and nondiseased arteries were used to detect the expressions of miR-10a/GATA6/VCAM-1 in pathogenic vs. normal conditions. In addition, sera from CAD patients and healthy subjects were collected to detect the level of circulating miR-10a. RESULTS: The comparison of human atherosclerotic coronary arteries with nondiseased arteries demonstrated that lower levels of endothelial miR-10a are related to human atherogenesis. Moreover, GATA6/VCAM-1 (a downstream target of miR-10a) was highly expressed in the endothelium, accompanied by the reduced levels of miR-10a during the development of human atherosclerosis. In addition, CAD patients had a significantly lower concentration of miR-10a in their serum compared to healthy subjects. CONCLUSIONS: Our findings suggest that low miR-10a and high GATA6/VCAM-1 in the cardiovascular endothelium correlates to the development of human atherosclerotic lesions, suggesting that miR-10a signaling has the potential to be developed as a biomarker for human atherosclerosis.

Punicalagin Attenuates Disturbed Flow-Induced Vascular Dysfunction by Inhibiting Force-Specific Activation of Smad1/5.

BACKGROUND: Pathophysiological vascular remodeling in response to disturbed flow with low and oscillatory shear stress (OSS) plays important roles in atherosclerosis progression. Pomegranate extraction (PE) was reported having anti-atherogenic effects. However, whether it can exert a beneficial effect against disturbed flow-induced pathophysiological vascular remodeling to inhibit atherosclerosis remains unclear. The present study aims at investigating the anti-atherogenic effects of pomegranate peel polyphenols (PPP) extraction and its purified compound punicalagin (PU), as well as their protective effects on disturbed flow-induced vascular dysfunction and their underlying molecular mechanisms. METHODS: The anti-atherogenic effects of PPP/PU were examined on low-density lipoprotein receptor knockout mice fed with a high fat diet. The vaso-protective effects of PPP/PU were examined in rat aortas using myograph assay. A combination of in vivo experiments on rats and in vitro flow system with human endothelial cells (ECs) was used to investigate the pharmacological actions of PPP/PU on EC dysfunction induced by disturbed flow. In addition, the effects of PPP/PU on vascular smooth muscle cell (VSMC) dysfunction were also examined. RESULTS: PU is the effective component in PPP against atherosclerosis. PPP/PU evoked endothelium-dependent relaxation in rat aortas. PPP/PU inhibited the activation of Smad1/5 in the EC layers at post-stenotic regions of rat aortas exposed to disturbed flow with OSS. PPP/PU suppressed OSS-induced expression of cell cycle regulatory and pro-inflammatory genes in ECs. Moreover, PPP/PU inhibited inflammation-induced VSMC dysfunction. CONCLUSION: PPP/PU protect against OSS-induced vascular remodeling through inhibiting force-specific activation of Smad1/5 in ECs and this mechanism contributes to their anti-atherogenic effects.

Mechanoresponsive Smad5 Enhances MiR-487a Processing to Promote Vascular Endothelial Proliferation in Response to Disturbed Flow.

MicroRNAs (miRs) and bone morphogenetic protein receptor-specific Smads are mechano-responsive molecules that play vital roles in modulating endothelial cell (EC) functions in response to blood flow. However, the roles of interplay between these molecules in modulating EC functions under flows remain unclear. We elucidated the regulatory roles of the interplay between miR-487a and Smad5 in EC proliferation in response to different flow patterns. Microarray and quantitative RT-PCR showed that disturbed flow with low and oscillatory shear stress (OS, 0.5 ± 4 dynes/cm2) upregulates EC miR-487a in comparison to static controls and pulsatile shear stress (12 ± 4 dynes/cm2). MiR-487a expression was higher in ECs in the inner curvature (OS region) than the outer curvature of the rat aortic arch and thoracic aorta and also elevated in diseased human coronary arteries. MiR-487a expression was promoted by nuclear phospho-Smad5, which bound to primary-miR-487a to facilitate miR-487a processing. Algorithm prediction and luciferase reporter and argonaute 2-immunoprecipitation assays demonstrated that miR-487a binds to 3'UTR of CREB binding protein (CBP) and p53. Knockdown and overexpression of miR-487a decreased and increased, respectively, phospho-Rb and cyclin A expressions through CBP and p53. A BrdU incorporation assay showed that miR-487a enhanced EC proliferation under OS in vitro and in disturbed flow regions of experimentally stenosed rat abdominal aorta in vivo. These results demonstrate that disturbed flow with OS induces EC expression of miR-487a through its enhanced processing by activated-Smad5. MiR-487 inhibits its direct targets CBP and p53 to induce EC cycle progression and proliferation. Our findings suggest that EC miR-487 may serve as an important molecular target for intervention against disturbed flow-associated vascular disorders resulting from atherosclerosis.

Effects of Chinese and Western Medicine on Patients with Dengue Fever.

Dengue fever is an important epidemic disease with a high prevalence in tropical and subtropical countries. We aimed to investigate the effects of a treatment integrating traditional Chinese (TCM) and Western medicines on dengue inpatients with warning signs (i.e., group B) according to the World Health Organization dengue classification in this retrospective cohort study of medical records. Inpatients who were treated with conventional Western therapies in the absence or presence of TCM were assigned to the control and treatment groups, respectively. Data were compared using an analysis of variance, general linear analysis, and chi-square test. The most common clinical symptoms and signs of dengue fever were fever and muscle ache. The treatment group patients were significantly more likely to present general weakness and poor appetite than the control group patients. Patients in the treatment group were more likely to experience stomachache than those in the control group. Moreover, comparisons of the changes in hemoglobin and alanine aminotransferase levels over time revealed significant differences between the patient groups. Zhu Ye Shi Gao Tang, Gui Pi Tang, Paeonia suffruticosa, and Clerodendrum cyrtophyllum were the most commonly administered TCM formula and single herbs in this study. Patients in the treatment group experienced a resolution of symptoms, signs, and laboratory data and were discharged smoothly, without deterioration to death or critical care. Our findings suggest that the integration of TCM and Western medicine may yield an appropriate treatment for dengue fever.

Induction of microRNA-10a using retinoic acid receptor-α and retinoid x receptor-α agonists inhibits atherosclerotic lesion formation.

BACKGROUND AND AIMS: MicroRNA (miR)-10a is a shear-regulated miR with the lowest expression in vascular endothelial cells (ECs) in athero-susceptible regions with oscillatory shear stress (OS). The aim of this study is to elucidate the relationship between EC miR-10a and atherosclerosis and develop a hemodynamics-based strategy for atherosclerosis treatment. METHODS: A combination of in vitro flow system and in vivo experimental animals was used to examine the functional roles of EC miR-10a and its clinical applications in atherosclerosis. RESULTS: En face staining showed that EC miR-10a is down-regulated in the inner curvature (OS region) of aortic arch in rats. Co-administration with retinoic acid receptor-α (RARα)- and retinoid X receptor-α (RXRα)-specific agonists rescued EC miR-10a expression in this OS region. These effects of OS and RARα/RXRα-specific agonists on EC miR-10a expression were confirmed by the in vitro flow system, and were modulated by the RARα-histone deacetylases complex, with the consequent modulation in the downstream GATA6/vascular cell adhesion molecule (VCAM)-1 signaling cascade. Animal studies showed that miR-10a levels are decreased in both aortic endothelium of atherosclerotic lesions and blood plasma from apolipoprotein E-deficient (ApoE-/-) mice. In vivo induction of EC miR-10a by administration of RARα/RXRα-specific agonists protects ApoE-/- mice from atherosclerosis through inhibition of GATA6/VCAM-1 signaling and inflammatory cell infiltration. CONCLUSIONS: Our findings indicate that down-regulation of miR-10a in aortic endothelium and blood serum is associated with atherosclerosis, and miR-10a has potential to be developed as diagnostic molecule for atherosclerosis. Moreover, EC miR-10a induction by RARα/RXRα-specific agonists is a potential hemodynamics-based strategy for atherosclerosis treatment.

Differential regulations of fibronectin and laminin in Smad2 activation in vascular endothelial cells in response to disturbed flow.

BACKGROUND: Atherosclerosis occurs in arterial curvatures and branches, where the flow is disturbed with low and oscillatory shear stress (OSS). The remodeling and alterations of extracellular matrices (ECMs) and their composition is the critical step in atherogenesis. In this study, we investigated the effects of different ECM proteins on the regulation of mechanotransduction in vascular endothelial cells (ECs) in response to OSS. METHODS: Through the experiments ranging from in vitro cell culture studies on effects of OSS on molecular signaling to in vivo examinations on clinical specimens from patients with coronary artery disease (CAD), we elucidated the roles of integrins and different ECMs, i.e., fibronectin (FN) and laminin (LM), in transforming growth factor (TGF)-ß receptor (TßR)-mediated Smad2 activation and nuclear factor-κB (NF-κB) signaling in ECs in response to OSS and hence atherogenesis. RESULTS: OSS at 0.5±12 dynes/cm2 induces sustained increases in the association of types I and II TßRs with ß1 and ß3 integrins in ECs grown on FN, but it only transient increases in ECs grown on LM. OSS induces a sustained activation of Smad2 in ECs on FN, but only a transient activation of Smad2 in ECs on LM. OSS-activation of Smad2 in ECs on FN regulates downstream NF-κB signaling and pro-inflammatory gene expression through the activation of ß1 integrin and its association with TßRs. In contrast, OSS induces transient activations of ß1 and ß3 integrins in ECs on LM, which associate with type I TßR to regulate Smad2 phosphorylation, resulting in transient induction of NF-κB and pro-inflammatory gene expression. In vivo investigations on diseased human coronary arteries from CAD patients revealed that Smad2 is highly activated in ECs of atherosclerotic lesions, which is accompanied by the concomitant increase of FN rather than LM in the EC layer and neointimal region of atherosclerotic lesions. CONCLUSIONS: Our findings provide new insights into the mechanisms of how OSS regulates Smad2 signaling and pro-inflammatory genes through the complex signaling networks of integrins, TßRs, and ECMs, thus illustrating the molecular basis of regional pro-inflammatory activation within disturbed flow regions in the arterial tree.

MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases.

Histone deacetylases (HDACs) and microRNAs (miRs) have emerged as two important epigenetic factors in the regulation of vascular physiology. This study aimed to elucidate the relationship between HDACs and miRs in the hemodynamic modulation of endothelial cell (EC) dysfunction. We found that miR-10a has the lowest expression among all examined shear-responsive miRs in ECs under oscillatory shear stress (OS), and a relatively high expression under pulsatile shear stress (PS). PS and OS alter EC miR-10a expression to regulate the expression of its direct target GATA6 and downstream vascular cell adhesion molecule (VCAM)-1. PS induces the expression, nuclear accumulation, and association of retinoid acid receptor-α (RARα) and retinoid X receptor-α (RXRα). RARα and RXRα serve as a "director" and an "enhancer," respectively, to enhance RARα binding to RA-responsive element (RARE) and hence miR-10a expression, thus down-regulating GATA6/VCAM-1 signaling in ECs. In contrast, OS induces associations of "repressors" HDAC-3/5/7 with RARα to inhibit the RARα-directed miR-10a signaling. The flow-mediated miR-10a expression is regulated by Krüppel-like factor 2 through modulation in RARα-RARE binding, with the consequent regulation in GATA6/VCAM-1 in ECs. These results are confirmed in vivo by en face staining on the aortic arch vs. the straight thoracic aorta of rats. Our findings identify a mechanism by which HDACs and RXRα modulate the hormone receptor RARα to switch miR-10a expression and hence the proinflammatory vs. anti-inflammatory responses of vascular endothelium under different hemodynamic forces.

MicroRNA mediation of endothelial inflammatory response to smooth muscle cells and its inhibition by atheroprotective shear stress.

RATIONALE: In atherosclerotic lesions, synthetic smooth muscle cells (sSMCs) induce aberrant microRNA (miR) profiles in endothelial cells (ECs) under flow stagnation. Increase in shear stress induces favorable miR modulation to mitigate sSMC-induced inflammation. OBJECTIVE: To address the role of miRs in sSMC-induced EC inflammation and its inhibition by shear stress. METHODS AND RESULTS: Coculturing ECs with sSMCs under static condition causes initial increases of 4 anti-inflammatory miRs (146a/708/451/98) in ECs followed by decreases below basal levels at 7 days; the increases for miR-146a/708 peaked at 24 hours and those for miR-451/98 lasted for only 6 to 12 hours. Shear stress (12 dynes/cm(2)) to cocultured ECs for 24 hours augments these 4 miR expressions. In vivo, these 4 miRs are highly expressed in neointimal ECs in injured arteries under physiological levels of flow, but not expressed under flow stagnation. MiR-146a, miR-708, miR-451, and miR-98 target interleukin-1 receptor-associated kinase, inhibitor of nuclear factor-κB kinase subunit-γ, interleukin-6 receptor, and conserved helix-loop-helix ubiquitous kinase, respectively, to inhibit nuclear factor-κB signaling, which exerts negative feedback control on the biogenesis of these miRs. Nuclear factor-E2-related factor (Nrf)-2 is critical for shear-induction of miR-146a in cocultured ECs. Silencing either Nrf-2 or miR-146a led to increased neointima formation of injured rat carotid artery under physiological levels of flow. Overexpressing miR-146a inhibits neointima formation of rat or mouse carotid artery induced by injury or flow cessation. CONCLUSIONS: Nrf-2-mediated miR-146a expression is augmented by atheroprotective shear stress in ECs adjacent to sSMCs to inhibit neointima formation of injured arteries.

Risk factors for Clostridium difficile-associated diarrhea among hospitalized adults with fecal toxigenic C. difficile colonization.

BACKGROUND: Patients with toxigenic Clostridium difficile colonization (tCDC) are at risk of developing C. difficile-associated diarrhea (CDAD). However, the risk factors of hospitalized patients with tCDC developing CDAD are not clear. METHODS: We conducted an 18-month prospective study at a medical ward in a district hospital in southern Taiwan. Within 48 hours of admission, weekly stool samples from asymptomatic hospitalized patients were obtained to detect fecal CDC. A polymerase chain reaction for tcdB was performed to determine toxigenic isolates. CDAD was diagnosed if the patient had diarrhea and toxigenic C. difficile present in a stool sample. RESULTS: A total 483 patients with stool samples were eligible for the study. Eighty-six (17.8%) patients had tCDC after screening, of whom 14 (16.3%) developed CDAD during follow-up. Among those with tCDC, patients with subsequent CDAD were more likely to have diabetes mellitus (p = 0.01) and to have received piperacillin-tazobactam (p = 0.04), or proton-pump inhibitors (PPIs; p = 0.04) than those without developing CDAD. The variables were statistically significant as determined by multivariate analysis. However, the 60-day crude mortality rates among tCDC patients with and without subsequent development of CDAD were similar. CONCLUSION: Diabetes mellitus and recent receipt of piperacillin-tazobactam or PPIs are independent risk factors for the development of CDAD among hospitalized patients with tCDC.

Risk factors of fecal toxigenic or non-toxigenic Clostridium difficile colonization: impact of Toll-like receptor polymorphisms and prior antibiotic exposure.

BACKGROUND: This study is to investigate the significance and risk factors of fecal toxigenic (tCdC) or non-toxigenic Clostridium difficile colonization (ntCdC) among hospitalized patients. METHODS: Adults admitted to medical wards in a district hospital between January 2011 and June 2012 were enrolled, and those with a history of colectomy, C. difficile fecal colonization or infection or receipt of either metronidazole or oral vancomycin within 3 months, were excluded. Stools collected within 48 hours after admission and every week during hospitalization were cultured for C. difficile. FINDINGS: Among the 441 enrolled patients, 84 (20.0%) had CdC at initial screening, including 58 (13.2%) with tCdC and 26 (6.8%) with ntCdC. Among patients with initial negative fecal screening for CdC, it took an average of 70.6 days or 66.5 days to develop tCdC or ntCdC during the study period. Finally 78 (17.7%) had tCdC and 34 (7.7%) had ntCdC. During the follow-up period, the patients with tCdC had a higher risk of CDAD (11/79, 14.1%) than those without CdC (3/328, 0.9%) and those with ntCdC (0/34, 0%) (P<0.001). In multivariate analysis, the TLR4 rs1927914 polymorphism (GG genotype) (odds ratio [OR] 4.4, 95% confidence interval [CI] 1.6-11.8, P = 0.003) and recent cefepime therapy (OR 5.3, 95% CI 2.1-13.2, P<0.001) were independently associated with tCdC, whereas recent cefuroxime (OR 11.7, 95% CI 2.3-60.2, P = 0.003) and glycopeptide therapy (OR 10.9, CI: 2.1-57.2, P = 0.005) associated with ntCdC. CONCLUSION: The incidence of CDAD is highest in patients with tCdC and lowest in patients with ntCdC, and the TLR4 rs1927914 polymorphism GG genotype and recent cefepime therapy were independently associated with tCdC.

Impact of toxigenic Clostridium difficile colonization and infection among hospitalized adults at a district hospital in southern Taiwan.

BACKGROUND: The impact of toxigenic Clostridium difficile colonization (tCDC) in hospitalized patients is not clear. AIM: To study the significance of tCDC in hospitalized patients. METHODS: A prospective study in the medical wards of a regional hospital was performed from January to June 2011. Fecal samples collected from patients at the time of admission were tested for tcdB by real-time polymerase chain reaction (PCR) and cultured for C. difficile. The patients were followed up weekly or when they developed diarrhea during hospitalization. If C. difficile was isolated, tcdA and tcdB would be tested by multiplex PCR. The primary outcome was the development of C. difficile-associated diarrhea (CDAD). FINDINGS: Of 168 patients enrolled, females predominated (87, 51.8%), and the mean patient age was 75.4 years old. Approximately 70% of the patients were nursing home residents, and one third had a recent hospitalization within the prior three months. Twenty-eight (16.7%) patients had tCDC, including 16 (9.5%) patients with tCDC at the time of admission and 12 (7.2%) with tCDC during the follow-up period. With regard to the medications taken during hospitalization, the patients were more likely to have tCDC if they had received more than one class of antibiotics than if they had received monotherapy (odds ratio [OR] 6.67, 95% confidence interval [CI] 1.41-31.56, P = 0.01), particularly if they received a glycopeptide in combination with a cephalosporin or penicillin or a cephalosporin and a carbapenem. More patients with tCDC developed CDAD than those without tCDC (17.9%, 5/28 vs. 1.4%, 2/140, P = 0.002). Overall 7 (4.2%) of the 168 patients developed CDAD, and crude mortality rate of those with and without tCDC was similar (21.4%, 6/28 vs. 19.4%, 27/140, P = 0.79). CONCLUSION: Recent use of glycopeptides and ß-lactam antibiotics is associated with toxigenic C. difficile colonization, which is a risk factor for developing C. difficile-associated diarrhea.

Convergence of physical and chemical signaling in the modulation of vascular smooth muscle cell cycle and proliferation by fibrillar collagen-regulated P66Shc.

Arterial smooth muscle cell (SMC) phenotype and proliferation is regulated by their surrounding collagens, which transform from fibrillar to monomeric type in atherogenesis, and platelet-derived growth factor (PDGF)-BB/interleukin (IL)-1ß. This study aims at elucidating the mechanisms by which physical (monomeric vs. fibrillar collagens) and chemical (PDGF-BB/IL-1ßvs. vehicle controls) stimuli modulate SMC cycle and proliferation. SMCs were cultured on monomeric vs. fibrillar type I collagens. In parallel experiments, SMCs on fibrillar collagen were co-stimulated with PDGF-BB/IL-1ß. These physical and chemical factors induced common SMC cycle signaling events, including up-regulations of cyclin-dependent kinase-4/6 and cyclins A/D1, phosphorylation of retinoblastoma (Rb) and its dissociations with E2F2/3. The physical and chemical inductions of SMC cycle signaling and progression were oppositely regulated by phosphatidylinositol 3-kinase (PI3K)-mediated Akt and p38 mitogen-activated protein kinase (MAPK). Fibrillar collagen degraded p66Shc, whose Ser36-phosphorylation plays important roles in the modulation of SMC cycle. Monomeric collagen and PDGF-BB/IL-1ß co-stimulation induced p66Shc expression and Ser36-phosphorylation through ß(1) integrin and PDGF receptor-ß, respectively. In conclusion, our results demonstrate that fibrillar collagen-regulated p66Shc converges the physical and chemical stimuli to modulate SMC cycle and proliferation through PI3K-mediated Akt and p38 MAPK and their opposite regulation in downstream common cell cycle signaling cascades.

Force-specific activation of Smad1/5 regulates vascular endothelial cell cycle progression in response to disturbed flow.

Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress, but the mechanism of force-specific activation of their signaling to modulate cellular function remains unclear. We have demonstrated that bone morphogenetic protein receptor (BMPR)-specific Smad1/5 can be force-specifically activated by oscillatory shear stress (OSS) in ECs to cause cell cycle progression. Smad1/5 is highly activated in ECs of atherosclerotic lesions in diseased human coronary arteries from patients with end-stage heart failure undergoing heart transplantation and from apolipoprotein E-deficient mice. Application of OSS (0.5 ± 4 dyn/cm(2)) causes the sustained activation of Smad1/5 in ECs through activations of mammalian target of rapamycin and p70S6 kinase, leading to up-regulation of cyclin A and down-regulations of p21(CIP1) and p27(KIP1) and, hence, EC cycle progression. En face examination of rat aortas reveals high levels of phospho-Smad1/5 in ECs of the inner, but not the outer, curvature of aortic arch, nor the straight segment of thoracic aorta [corrected]. Immunohistochemical and en face examinations of the experimentally stenosed abdominal aorta in rats show high levels of phospho-Smad1/5 in ECs at poststenotic sites, where OSS occurs. These OSS activations of EC Smad1/5 in vitro and in vivo are not inhibited by the BMP-specific antagonist Noggin and, hence, are independent of BMP ligand. Transfecting ECs with Smad1/5-specific small interfering RNAs inhibits the OSS-induced EC cycle progression. Our findings demonstrate the force-specificity of the activation of Smad1/5 and its contribution to cell cycle progression in ECs induced by disturbed flow.

Role of histone deacetylases in transcription factor regulation and cell cycle modulation in endothelial cells in response to disturbed flow.

Vascular endothelial cells (ECs) are exposed to different flow patterns (i.e., disturbed vs. laminar), and the associated oscillatory shear stress (OSS) or pulsatile shear stress (PSS) lead to differential responses. We investigated the roles of class I and II histone deacetylases (HDAC-1/2/3 and HDAC-5/7, respectively) in regulating NF-E2-related factor-2 (Nrf2) and Krüppel-like factor-2 (KLF2), two transcription factors governing many shear-responsive genes, and the cell cycle in ECs in response to OSS. Application of OSS (0.5 ± 4 dynes/cm(2)) to cultured ECs sustainably up-regulated class I and II HDACs and their nuclear accumulation, whereas PSS (12 ± 4 dynes/cm(2)) induced phosphorylation-dependent nuclear export of class II HDACs. En face immunohistochemical examination of rat aortic arch and experimentally stenosed abdominal aorta revealed high HDAC-2/3/5 levels in ECs in areas exposed to disturbed flow. OSS induced the association of HDAC-1/2/3 with Nrf2 and HDAC-3/5/7 with myocyte enhancer factor-2; deacetylation of these factors led to down-regulation of antioxidant gene NAD(P)H quinone oxidoreductase-1 (NQO1) and KLF2. HDAC-1/2/3- and HDAC-3/5/7-specific small interfering RNAs eliminated the OSS-induced down-regulation of NQO1 and KLF2, respectively. OSS up-regulated cyclin A and down-regulated p21(CIP1) in ECs and induced their proliferation; these effects were mediated by HDAC-1/2/3. Intraperitoneal administration of the class I-specific HDAC inhibitor valproic acid into bromodeoxyuridine (BrdU)-infused rats inhibited the increased EC uptake of BrdU at poststenotic sites. The OSS-induced HDAC signaling and EC responses are mediated by phosphatidylinositol 3-kinase/Akt. Our findings demonstrate the important roles of different groups of HDACs in regulating the oxidative, inflammatory, and proliferative responses of ECs to disturbed flow with OSS.

Estrogen augments shear stress-induced signaling and gene expression in osteoblast-like cells via estrogen receptor-mediated expression of beta1-integrin.

Estrogen and mechanical forces are positive regulators for osteoblast proliferation and bone formation. We investigated the synergistic effect of estrogen and flow-induced shear stress on signal transduction and gene expression in human osetoblast-like MG63 cells and primary osteoblasts (HOBs) using activations of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and expressions of c-fos and cyclooxygenase-2 (I) as readouts. Estrogen (17beta-estradiol, 10 nM) and shear stress (12 dyn/cm(2)) alone induced transient phosphorylations of ERK and p38 MAPK in MG63 cells. Pretreating MG63 cells with 17beta-estradiol for 6 hours before shearing augmented these shear-induced MAPK phosphorylations. Western blot and flow cytometric analyses showed that treating MG63 cells with 17beta-estradiol for 6 hrs induced their beta(1)-integrin expression. This estrogen-induction of beta(1)-integrin was inhibited by pretreating the cells with a specific antagonist of estrogen receptor ICI 182,780. Both 17beta-estradiol and shear stress alone induced c-fos and Cox-2 gene expressions in MG63 cells. Pretreating MG63 cells with 17beta-estradiol for 6 hrs augmented the shear-induced c-fos and Cox-2 expressions. The augmented effects of 17beta-estradiol on shear-induced MAPK phosphorylations and c-fos and Cox-2 expressions were inhibited by pretreating the cells with ICI 182,780 or transfecting the cells with beta(1)-specific small interfering RNA. Similar results on the augmented effect of estrogen on shear-induced signaling and gene expression were obtained with HOBs. Our findings provide insights into the mechanism by which estrogen augments shear stress responsiveness of signal transduction and gene expression in bone cells via estrogen receptor-mediated increases in beta(1)-integrin expression.

Extended-spectrum beta-lactamase-producing phenotype signifies a poor prognosis for patients with cefpodoxime-resistant Escherichia coli or Klebsiella pneumoniae bacteremia.

BACKGROUND AND PURPOSE: Bloodstream infections caused by multidrug-resistant Enterobacteriaceae are a major concern. This study explored the clinical impact of extended-spectrum beta-lactamase (ESBL) production among cefpodoxime-resistant Escherichia coli and Klebsiella pneumoniae bacteremia. METHODS: The medical charts and microbiological results of patients with cefpodoxime-resistant E. coli or K. pneumoniae bacteremia in a tertiary hospital in southern Taiwan between June 2003 and December 2006 were retrospectively reviewed. The clinical characteristics, medical histories, and clinical outcomes were evaluated. ESBL production was indicated by the double-disk synergy test. RESULTS: 278 episodes of bacteremia caused by cefpodoxime-resistant K. pneumoniae or E. coli were identified, of which 115 (41%) were ESBL producing. Compared with non-ESBL-producing bacteremia, bacteremic episodes caused by ESBL producers were less often community acquired (4.3% vs 26.4%; p < 0.001). Underlying diabetes mellitus (48.7% vs 35.0%; p = 0.02), liver cirrhosis (22.6% vs 11.7%; p = 0.02), or uremia (21.7% vs 3.7%; p < 0.001) were more common in ESBL-producing bacteremia. In contrast, solid tumors were more frequent in non-ESBL-producing bacteremia (44.8% vs 27.8%; p = 0.004). Overall, patients with ESBL-producing bacteremia had higher disease severity indicated by a Pittsburgh bacteremia score > or = 4, longer duration of hospital stay (51.1 days vs 31.9 days; p = 0.007), more admission to intensive care units (19.1% vs 8.0%; p = 0.006), and a higher mortality rate at 28 days (34.8% vs 23.9%; p = 0.03). CONCLUSIONS: ESBL production signifies a poor clinical outcome for patients with bacteremia caused by cefpodoxime-resistant E. coli or K. pneumoniae.

Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress.

E-selectin is a major adhesion molecule expressed by endothelial cells (ECs), which are exposed to shear stress and neighboring smooth muscle cells (SMCs). We investigated the mechanisms underlying the modulation of EC E-selectin expression by SMCs and shear stress. SMC coculture induced rapid and sustained increases in expression of E-selectin and phosphorylation of interleukin-1 (IL-1) receptor-associated kinase glycoprotein-130, as well as the downstream mitogen-activated protein kinases (MAPKs) and Akt. By using specific inhibitors, dominant-negative mutants, and small interfering RNA, we demonstrated that activations of c-Jun-NH(2)-terminal kinase (JNK) and p38 of the MAPK pathways are critical for the coculture-induced E-selectin expression. Gel shifting and chromatin immunoprecipitation assays showed that SMC coculture increased the nuclear factor-kappaB (NF-kappaB)-promoter binding activity in ECs; inhibition of NF-kappaB activation by p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced E-selectin promoter activity. Protein arrays and blocking assays using neutralizing antibodies demonstrated that IL-1beta and IL-6 produced by EC/SMC cocultures are major contributors to the coculture induction of EC signaling and E-selectin expression. Preshearing of ECs at 12 dynes/cm(2) inhibited the coculture-induced EC signaling and E-selectin expression. Our findings have elucidated the molecular mechanisms underlying the SMC induction of EC E-selectin expression and the shear stress protection against this SMC induction.