Essential role for EGFR tyrosine kinase and ER stress in myocardial infarction in type 2 diabetes.

We previously reported that EGFR tyrosine kinase (EGFRtk) activity and endoplasmic reticulum (ER) stress are enhanced in type 2 diabetic (T2D) mice and cause vascular dysfunction. In the present study, we determined the in vivo contribution of EGFRtk and ER stress in acute myocardial infarction induced by acute ischemia (40 min)-reperfusion (24 h) (I/R) injury in T2D (db-/db-) mice. We treated db-/db- mice with EGFRtk inhibitor (AG1478, 10 mg/kg/day) for 2 weeks. Mice were then subjected to myocardial I/R injury. The db-/db- mice developed a significant infarct after I/R injury. The inhibition of EGFRtk significantly reduced the infarct size and ER stress induction. We also determined that the inhibition of ER stress (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day) in db-/db- significantly decrease the infarct size indicating that ER stress is a downstream mechanism to EGFRtk. Moreover, AG1478 and TUDCA reduced myocardium p38 and ERK1/2 MAP-kinases activity, and increased the activity of the pro-survival signaling cascade Akt. Additionally, the inhibition of EGFRtk and ER stress reduced cell apoptosis and the inflammation as indicated by the reduction in macrophages and neutrophil infiltration. We determined for the first time that the inhibition of EGFRtk protects T2D heart against I/R injury through ER stress-dependent mechanism. The cardioprotective effect of EGFRtk and ER stress inhibition involves the activation of survival pathway, and inhibition of apoptosis, and inflammation. Thus, targeting EGFRtk and ER stress has the potential for therapy to overcome myocardial infarction in T2D.

Essential Role of IL-12 in Angiogenesis in Type 2 Diabetes.

Recently, IL-12 emerged as a critical player in type 2 diabetes complications. We previously reported that ischemia-induced angiogenesis is compromised in type 2 diabetic mice. In this study, we determined that IL-12 disruption rescued angiogenesis and arteriogenesis in type 2 diabetic mice. To induce type 2 diabetes, wild-type (WT), p40IL-12-/- (p40-/-), and p35IL-12-/- (p35-/-) mice were fed a high-fat diet (HFD) for 12 weeks. Body weight, glucose test tolerance, and insulin test tolerance were assessed. After 12 weeks of an HFD, the femoral artery was ligated and blood flow recovery was measured every week for 4 weeks. WT, p40-/-, and p35-/- mice fed an HFD become obese after 12 weeks and exhibit glucose intolerance and insulin resistance. Blood flow recovery was fully restored in 2 to 3 weeks after femoral artery ligation in all groups of mice fed a normal diet. However, after 12 weeks of an HFD, blood flow recovery was compromised in WT mice, whereas it was fully recovered in p40-/- and p35-/- mice. The mechanism of blood flow recovery involves an increase in capillary/arteriole density, endothelial nitric oxide synthase/Akt/vascular endothelial growth factor receptor 2 signaling, and a reduction in oxidative stress and inflammation. The disruption of IL-12 promotes angiogenesis and increases blood flow recovery in obese type 2 diabetic mice by an endothelial nitric oxide synthase/Akt/vascular endothelial growth factor receptor 2/oxidative stress-inflammation-dependent mechanism.

Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function.

Endothelial barrier function is tightly regulated by plasma membrane receptors and is crucial for tissue fluid homeostasis; its dysfunction causes disease, including sepsis and inflammation. The ubiquitous activation of Ca2+ signaling upon phospholipase C-coupled receptor ligation leads quite naturally to the assumption that Ca2+ signaling is required for receptor-regulated endothelial barrier function. This widespread hypothesis draws analogy from smooth muscle and proposes the requirement of G protein-coupled receptor (GPCR)-generated Ca2+ signaling in activating the endothelial contractile apparatus and generating interendothelial gaps. Notwithstanding endothelia being non-excitable in nature, the hypothesis of Ca2+-induced endothelial contraction has been invoked to explain actions of GPCR agonists that either disrupt or stabilize endothelial barrier function. Here, we challenge this correlative hypothesis by showing a lack of causal link between GPCR-generated Ca2+ signaling and changes in human microvascular endothelial barrier function. We used three endogenous GPCR agonists: thrombin and histamine, which disrupt endothelial barrier function, and sphingosine-1-phosphate, which stabilizes barrier function. The qualitatively different effects of these three agonists on endothelial barrier function occur independently of Ca2+ entry through the ubiquitous store-operated Ca2+ entry channel Orai1, global Ca2+ entry across the plasma membrane, and Ca2+ release from internal stores. However, disruption of endothelial barrier function by thrombin and histamine requires the Ca2+ sensor stromal interacting molecule-1 (STIM1), whereas sphingosine-1-phosphate-mediated enhancement of endothelial barrier function occurs independently of STIM1. We conclude that although STIM1 is required for GPCR-mediated disruption of barrier function, a causal link between GPCR-induced cytoplasmic Ca2+ increases and acute changes in barrier function is missing. Thus, the cytosolic Ca2+-induced endothelial contraction is a cum hoc fallacy that should be abandoned.

Essential Role of Smooth Muscle STIM1 in Hypertension and Cardiovascular Dysfunction.

OBJECTIVES: Chronic hypertension is the most critical risk factor for cardiovascular disease, heart failure, and stroke. APPROACH AND RESULTS: Here we show that wild-type mice infused with angiotensin II develop hypertension, cardiac hypertrophy, perivascular fibrosis, and endothelial dysfunction with enhanced stromal interaction molecule 1 (STIM1) expression in heart and vessels. All these pathologies were significantly blunted in mice lacking STIM1 specifically in smooth muscle (Stim1(SMC-/-)). Mechanistically, STIM1 upregulation during angiotensin II-induced hypertension was associated with enhanced endoplasmic reticulum stress, and smooth muscle STIM1 was required for endoplasmic reticulum stress-induced vascular dysfunction through transforming growth factor-ß and nicotinamide adenine dinucleotide phosphate oxidase-dependent pathways. Accordingly, knockout mice for the endoplasmic reticulum stress proapoptotic transcriptional factor, CCAAT-enhancer-binding protein homologous protein (CHOP(-/-)), were resistant to hypertension-induced cardiovascular pathologies. Wild-type mice infused with angiotensin II, but not Stim1(SMC-/-) or CHOP(-/-) mice showed elevated vascular nicotinamide adenine dinucleotide phosphate oxidase activity and reduced phosphorylated endothelial nitric oxide synthase, cGMP, and nitrite levels. CONCLUSIONS: Thus, smooth muscle STIM1 plays a crucial role in the development of hypertension and associated cardiovascular pathologies and represents a promising target for cardiovascular therapy.

Leukotriene-C4 synthase, a critical enzyme in the activation of store-independent Orai1/Orai3 channels, is required for neointimal hyperplasia.

Leukotriene-C4 synthase (LTC4S) generates LTC4 from arachidonic acid metabolism. LTC4 is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC4 was also a cytosolic second messenger that activated store-independent LTC4-regulated Ca(2+) (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC4S in neointima formation remains unknown. Here we show that LTC4S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC4S protein expression in VSMCs. Inhibition of LTC4S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC4S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC4S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC4S or Orai3 was prevented by shRNA. We conclude that LTC4S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.

Augmented EGF receptor tyrosine kinase activity impairs vascular function by NADPH oxidase-dependent mechanism in type 2 diabetic mouse.

We previously determined that augmented EGFR tyrosine kinase (EGFRtk) impairs vascular function in type 2 diabetic mouse (TD2). Here we determined that EGFRtk causes vascular dysfunction through NADPH oxidase activity in TD2. Mesenteric resistance arteries (MRA) from C57/BL6 and db-/db- mice were mounted in a wired myograph and pre-incubated for 1h with either EGFRtk inhibitor (AG1478) or exogenous EGF. The inhibition of EGFRtk did not affect the contractile response to phenylephrine-(PE) and thromboxane-(U46619) or endothelium-dependent relaxation (EDR) to acetylcholine in MRA from control group. However, in TD2 mice, AG1478 reduced the contractile response to U46619, improved vasodilatation and reduced p22phox-NADPH expression, but had no effect on the contractile response to PE. The incubation of MRA with exogenous EGF potentiated the contractile response to PE in MRA from control and diabetic mice. However, EGF impaired the EDR and potentiated the vasoconstriction to U46619 only in the control group. Interestingly, NADPH oxidase inhibition in the presence of EGF restored the normal contraction to PE and improved the EDR but had no effect on the potentiated contraction to U46619. Vascular function improvement was associated with the rescue of eNOS and Akt and reduction in phosphorylated Rho-kinase, NOX4 mRNA levels, and NADPH oxidase activity. MRA from p47phox-/- mice incubated with EGF potentiated the contraction to U46619 but had no effect to PE or ACh responses. The present study provides evidence that augmented EGFRtk impairs vascular function by NADPH oxidase-dependent mechanism. Therefore, EGFRtk and oxidative stress should be potential targets to treat vascular dysfunction in TD2.

Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction.

BACKGROUND: We recently reported that ER stress plays a key role in vascular endothelial dysfunction during hypertension. In this study we aimed to elucidate the mechanisms by which ER stress induction and oxidative stress impair vascular endothelial function. METHODOLOGY/PRINCIPAL FINDINGS: We conducted in vitro studies with primary endothelial cells from coronary arteries stimulated with tunicamycin, 1µg/mL, in the presence or absence of two ER stress inhibitors: tauroursodeoxycholic acid (Tudca), 500µg/mL, and 4-phenylbutyric acid (PBA), 5mM. ER stress induction was assessed by enhanced phosphorylation of PERK and eIF2α, and increased expression of CHOP, ATF6 and Grp78/Bip. The ER stress induction increased p38 MAPK phosphorylation, Nox2/4 mRNA levels and NADPH oxidase activity, and decreased eNOS promoter activity, eNOS expression and phosphorylation, and nitrite levels. Interestingly, the inhibition of p38 MAPK pathway reduced CHOP and Bip expressions enhanced by tunicamycin and restored eNOS promoter activation as well as phosphorylation. To study the effects of ER stress induction in vivo, we used C57BL/6J mice and p47phox(-/-) mice injected with tunicamycin or saline. The ER stress induction in mice significantly impaired vascular endothelium-dependent and independent relaxation in C57BL/6J mice compared with p47phox(-/-) mice indicating NADPH oxidase activity as an intermediate for ER stress in vascular endothelial dysfunction. CONCLUSION/SIGNIFICANCE: We conclude that chemically induced ER stress leads to a downstream enhancement of p38 MAPK and oxidative stress causing vascular endothelial dysfunction. Our results indicate that inhibition of ER stress could be a novel therapeutic strategy to attenuate vascular dysfunction during cardiovascular diseases.

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.

The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.

Differential role for stromal interacting molecule 1 in the regulation of vascular function.

We determined the in vivo role of stromal-interacting molecule 1 (STIM1) in the regulation of vascular function using endothelial cell (EC)- and smooth-muscle (SM)-specific knockout mice. Systolic blood pressure and glucose levels were similar in all mice (Stim1(SMC-/-), Stim1(SMC-/+), Stim1(EC-/-), Stim1(EC-/+)), but body weight was reduced in Stim1(EC-/-) and Stim1(SMC-/-) mice. The contraction of arteries in response to phenylephrine was significantly reduced in Stim1(SMC-/-) mice only. However, contraction to thromboxane and KCl was similar in all groups. The endothelium-dependent relaxation (EDR) was impaired in Stim1(EC-/+) and drastically reduced in Stim1(EC-/-) mice while the endothelium-independent vasorelaxation was similar among all groups. Acute downregulation of STIM1 in arteries reduced EDR and the contractile response to phenylephrine, while the contractile response to thromboxane was not affected. NADPH oxidase activity was increased only in Stim1(EC-/+) and Stim1(EC-/-) mice. Calcium (Ca(2+)) entry in endothelial cells stimulated with thrombin and histamine had the pharmacological features of store-operated Ca(2+) entry (SOCE) and was dependent on STIM1 expression. We conclude that STIM1 plays opposing roles in vascular smooth muscle vs. endothelial cells in the regulation of vascular reactivity.

Nuclear factor kappa B inhibition improves conductance artery function in type 2 diabetic mice.

BACKGROUND: We previously reported that enhanced nuclear factor kappa B (NFκB) activity is responsible for resistance arteries dysfunction in type 2 diabetic mice. METHODS: In this study, we aimed to determine whether augmented NFκB activity also impairs conductance artery (thoracic aorta) function in type 2 diabetic mice. We treated type 2 diabetic (db(-) /db(-) ) and control (db(-) /db(+) ) mice with two NFκB inhibitors (dehydroxymethylepoxyquinomicin, 6 mg/kg, twice a week and IKK-NBD peptide, 500 µg/kg/day) for 4 weeks. RESULTS: As expected, the NFκB inhibition did not affect blood glucose level and body weight. Thoracic aorta vascular endothelium-dependent relaxation (EDR), determined by the wire myograph, was impaired in diabetic mice compared with control and was significantly improved after NFκB inhibition. Interestingly, thoracic EDR was also rescued in db(-) /db(-p50NFκB-/-) and db(-) /db(-PARP-1-/-) double knockout mice compared with db(-) /db(-) mice. Similarly, the acute in vitro down regulation of NFκB-p65 using p65 shRNA lentiviral particles in arteries from db(-) /db(-) mice also improved thoracic aorta EDR. Western blot analysis showed that the p65NFκB phosphorylation, cleaved PARP-1 and COX-2 expression were increased in thoracic aorta from diabetic mice, which were restored after NFκB inhibition and in db(-) /db(-p-50NFκB-/-) and db(-) /db(-PARP-1-/-) mice. CONCLUSIONS: The present results indicate that in male type 2 diabetic mice, the augmented NFκB activity also impairs conductance artery function through PARP-1 and COX-2-dependent mechanisms.

Store-independent Orai1/3 channels activated by intracrine leukotriene C4: role in neointimal hyperplasia.

RATIONALE: Through largely unknown mechanisms, Ca(2+) signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca(2+) entry has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca(2+) entry pathways, its upregulation during VSMC remodeling, and its contribution to neointima formation remain unknown. OBJECTIVE: The goal of this study was to determine the agonist-evoked Ca(2+) entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon injury of rat carotid arteries. METHODS AND RESULTS: Ca(2+) imaging and patch-clamp recordings showed that although the platelet-derived growth factor activates the canonical Ca(2+) release-activated Ca(2+) channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca(2+)-selective channel contributed by Orai1, Orai3, and stromal interacting molecule1 in the same cells. Unexpectedly, Ca(2+) store depletion is not required for activation of Orai1/3 channel by thrombin. Rather, the signal for Orai1/3 channel activation is cytosolic leukotrieneC4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC4 synthase. Importantly, Orai3 is upregulated in an animal model of VSMC neointimal remodeling, and in vivo Orai3 knockdown inhibits neointima formation. CONCLUSIONS: These results demonstrate that distinct native Ca(2+)-selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC4 acts through hitherto unknown intracrine mode to elicit store-independent Ca(2+) signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

Enhanced p22phox expression impairs vascular function through p38 and ERK1/2 MAP kinase-dependent mechanisms in type 2 diabetic mice.

Type 2 diabetes is associated with vascular complication. We hypothesized that increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p22(phox) expression impairs vascular endothelium-dependent relaxation (EDR) in type 2 diabetes. Type 2 diabetic (db(-)/db(-)) and control (db(-)/db(+)) mice were treated with reactive oxygen species (ROS) scavenger, polyethylene glycol superoxide dismutase (1,000 U/kg daily ip), or small interfering RNA p22(phox) (p22(phox)-lentivirus-small interfering RNA, 100 µg iv, 2 times/wk) for 1 mo. EDR was impaired in microvascular bed (coronary arteriole and femoral and mesenteric resistance arteries) from diabetic mice compared with control. Interestingly, ROS scavenger and p22(phox) downregulation did not affect blood glucose level or body weight but significantly improved EDR. Mitogen-activated protein kinases (ERK1/2 and p38) phosphorylation and NADPH oxidase activity were increased in arteries from diabetic mice and were reduced after ROS scavenger or p22(phox) downregulation in db(-)/db(-) mice. The present study showed that enhanced p22(phox) expression causes vascular dysfunction through ERK1/2 and p38-mitogen-activated protein kinase-dependent mechanisms in male type 2 diabetic mice. Therefore, p22(phox) could be an important target to improve vascular function in diabetes.

Vasodilator responses to acetylcholine are not mediated by the activation of soluble guanylate cyclase or TRPV4 channels in the rat.

The effects of 1H-[1,2,4]-oxadizaolo[4,3-]quinoxaline-1-one (ODQ), an inhibitor of the activation of soluble guanylate cyclase (sGC) on responses to NO donors acetylcholine (ACh) and bradykinin (BK) were investigated in the pulmonary and systemic vascular beds of the rat. In these studies the administration of ODQ in a dose of 5 mg/kg iv attenuated vasodilator responses to five different NO donors without inhibiting responses to ACh and BK in the systemic and pulmonary vascular beds of the rat. Vasodilator responses to ACh were not inhibited by l-NAME or the transient receptor vanilloid type 4 (TRPV4) antagonist GSK-2193874, which attenuated vasodilator responses to the TRPV4 agonist GSK-1016790A. ODQ did not inhibit vasodilator responses to agents reported to act in an NO-independent manner or to vasoconstrictor agents, and ODQ did not increase blood methemoglobin levels, suggesting that off target effects were minimal. These results show that ODQ in a dose that inhibited NO donor-mediated responses did not alter vasodilator responses to ACh in the pulmonary and systemic vascular beds and did not alter systemic vasodilator responses to BK. The present results indicate that decreases in pulmonary and systemic arterial pressures in response to ACh are not mediated by the activation of sGC or TRPV4 channels and that ODQ can be used to study the role of the activation of sGC in mediating vasodilator responses in the rat.

Orai3 is an estrogen receptor α-regulated Ca²âº channel that promotes tumorigenesis.

Store-operated Ca(2+) entry (SOCE) encoded by Orai1 proteins is a ubiquitous Ca(2+)-selective conductance involved in cellular proliferation and migration. We recently described up-regulation of Orai3 channels that selectively mediate SOCE in estrogen receptor α-expressing (ERα(+)) breast cancer cells. However, the connection between ERα and Orai3 and the role of Orai3 in tumorigenesis remain unknown. Here, we show that ERα knockdown decreases Orai3 mRNA (by ∼63%) and protein (by ∼44%) with no effect on Orai1. ERα knockdown decreases Orai3-mediated SOCE (by ∼43%) and the corresponding Ca(2+) release-activated Ca(2+) (CRAC) current (by ∼42%) in ERα(+) MCF7 cells. The abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of Orai3. ERα activation increased Orai3 expression and SOCE in MCF7 cells. Epidermal growth factor (EGF) and thrombin stimulate Ca(2+) influx into MCF7 cells through Orai3. Orai3 knockdown inhibited SOCE-dependent phosphorylation of extracellular signal-regulated kinase (ERK1/2; by ∼44%) and focal adhesion kinase (FAK; by ∼46%) as well as transcriptional activity of nuclear factor for activated T cells (NFAT; by ∼49%). Significantly, Orai3 knockdown selectively decreased anchorage-independent growth (by ∼58%) and Matrigel invasion (by ∼44%) of ERα(+) MCF7 cells with no effect on ERα(-) MDA-MB231 cells. Moreover, Orai3 knockdown inhibited ERα(+) cell tumorigenesis in immunodeficient mice (∼66% reduction in tumor volume). These data establish Orai3 as an ERα-regulated channel and a potential selective therapeutic target for ERα(+) breast cancers.

Ac-SDKP attenuates ER stress-stimulated collagen production in cardiac fibroblasts by inhibiting CHOP-mediated NF-κB expression.

Inflammation and cardiac fibrosis are prevalent pathophysiologic conditions associated with hypertension, cardiac remodeling, and heart failure. Endoplasmic reticulum (ER) stress triggers the cells to activate unfolded protein responses (UPRs) and upregulate the ER stress chaperon, enzymes, and downstream transcription factors to restore normal ER function. The mechanisms that link ER stress-induced UPRs upregulation and NF-κB activation that results in cardiac inflammation and collagen production remain elusive. N-Acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a natural tetrapeptide that negatively regulates inflammation and fibrosis, has been reported. Whether it can inhibit ER stress-induced collagen production in cardiac fibroblasts remains unclear. Thus, we hypothesized that Ac-SDKP attenuates ER stress-stimulated collagen production in cardiac fibroblasts by inhibiting CHOP-mediated NF-κB expression. We aimed to study whether Ac-SDKP inhibits tunicamycin (TM)-induced ER stress signaling, NF-κB signaling, the release of inflammatory cytokine interleukin-6, and collagen production in human cardiac fibroblasts (HCFs). HCFs were pre-treated with Ac-SDKP (10 nM) and then stimulated with TM (0.25 µg/mL). We found that Ac-SDKP inhibits TM-induced collagen production by attenuating ER stress-induced UPRs upregulation and CHOP/NF-κB transcriptional signaling pathways. CHOP deletion by specific shRNA maintains the inhibitory effect of Ac-SDKP on NF-κB and type-1 collagen (Col-1) expression at both protein and mRNA levels. Attenuating ER stress-induced UPR sensor signaling by Ac-SDKP seems a promising therapeutic strategy to combat detrimental cardiac inflammation and fibrosis.

Role of plasmacytoid dendritic cells in vascular dysfunction in mice with renovascular hypertension.

Endothelial dysfunction and inflammation are clinically significant risk factors for cardiovascular diseases in hypertension. Although immune cells play a role in hypertension, the impact of plasmacytoid dendritic cells in established renovascular hypertension-induced cardiovascular complications is not fully understood. We investigated plasmacytoid dendritic cells' contribution to arterial endothelial dysfunction and inflammation in renovascular hypertension. A two-kidney one-clip (2K1C) model for four weeks in both male and female mice was used to induce renovascular hypertension. We treated mice with or without anti-PDCA-1 antibodies for one week to deplete the plasmacytoid dendritic cells. Renovascular hypertension causes cardiac hypertrophy, lung edema, and microvascular endothelial dysfunction associated with inflammation induction in mice. Moreover, renovascular hypertension affects the profile of immune cells, including dendritic cells and macrophages, with variations between male and female mice. Interestingly, the depletion of plasmacytoid dendritic cells significantly reduces blood pressure, cardiac hypertrophy, lung edema, inflammation, and oxidative stress and improves microvascular endothelial function via the endoplasmic reticulum (ER) stress, autophagy, and mTOR-dependent mechanisms. Plasmacytoid dendritic cells significantly contribute to the development of cardiovascular complications in renovascular hypertension by modulating immune cells, inflammation, oxidative stress, and ER stress.

STIM1 and Orai1 mediate CRAC channel activity and are essential for human glioblastoma invasion.

The Ca(2+) sensor stromal interacting molecule 1 (STIM1) and the Ca(2+) channel Orai1 mediate the ubiquitous store-operated Ca(2+) entry (SOCE) pathway activated by depletion of internal Ca(2+) stores and mediated through the highly Ca(2+)-selective, Ca(2+) release-activated Ca(2+) (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca(2+) currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca(2+) imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.

Chronic inhibition of epidermal growth factor receptor tyrosine kinase and extracellular signal-regulated kinases 1 and 2 (ERK1/2) augments vascular response to limb ischemia in type 2 diabetic mice.

Type 2 diabetes is a key risk factor for ischemia-dependent pathology; therefore, a significant medical need exists to develop novel therapies that increase the formation of new vessels. We explored the therapeutic potential of epidermal growth factor receptor tyrosine kinase (EGFRtk) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibition in impaired ischemia-induced neovascularization in type 2 diabetes. Unilateral femoral artery ligation was performed in diabetic (db(-)/db(-)) and their control (db(-)/db(+)) mice for 4 weeks, followed by treatments with EGFRtk and ERK1/2 inhibitors (AG1478, 10 mg/kg/day and U0126, 400 µg/kg/day, respectively) for 3 weeks. Neovascularization, blood flow recovery, vascular and capillary density, and endothelial nitric oxide synthase activity were significantly impaired and were associated with enhanced EGFRtk and ERK1/2 activity in db(-)/db(-) mice. EGFRtk and ERK1/2 inhibitors did not have any effect in control mice, while in db(-)/db(-) mice there was a significant increase in neovascularization, blood flow recovery, vascular and capillary density, endothelial nitric oxide synthase activity, and were associated with a decrease in EGFRtk and ERK1/2 activity. Our data demonstrated that the inhibition of EGFRtk and ERK1/2 restored ischemia-induced neovascularization and blood flow recovery in type 2 diabetic mice. Thus, EGFRtk and ERK1/2 could be possible targets to protect from ischemia-induced vascular pathology in type 2 diabetes.