Anna Alexanian

Acute Exposure to Low Glucose Rapidly Induces Endothelial Dysfunction and Mitochondrial Oxidative Stress Role for AMP Kinase

Objective—Hypoglycemia is associated with increased mortality. The reasons for this remain unclear, and the effects of low glucose exposure on vascular endothelial function remain largely unknown. We endeavored to determine the effects of low glucose on endothelial cells and intact human arterioles. Methods and Results—We exposed human umbilical vein endothelial cells to low glucose conditions in a clinically relevant range (40–70 mg/dL) and found rapid and marked reductions in nitric oxide (NO) bioavailability (P<0.001). This was associated with concomitantly increased mitochondrial superoxide production (P<0.001) and NO-dependent mitochondrial hyperpolarization (P<0.001). Reduced NO bioavailability was rapid and attributable to reduced endothelial nitric oxide synthase activity and destruction of NO. Low glucose rapidly activated AMP kinase, but physiological activation failed to restore NO bioavailability. Pharmacological AMP kinase activation led to phosphorylation of endothelial nitric oxide synthases Ser633 activation site, reversing the adverse effects of low glucose. This protective effect was prevented by L-NG-Nitroarginine methyl ester. Intact human arterioles exposed to low glucose demonstrated marked endothelial dysfunction, which was prevented by either metformin or TEMPOL. Conclusion—Our data suggest that moderate low glucose exposure rapidly impairs NO bioavailability and endothelial function in the human endothelium and that pharmacological AMP kinase activation inhibit this effect in an NO-dependent manner.

Pyk2 mediates endothelin-1 signaling via p130Cas/BCAR3 cascade and regulates human glomerular mesangial cell adhesion and spreading

Calcium‐regulated non‐receptor proline‐rich tyrosine kinase 2 (Pyk2) is a critical mediator of endothelin‐1 (ET‐1) signaling in human glomerular mesangial cells (GMC). We aimed to identify which small G‐protein is acting downstream of Pyk2. Dominant interfering Pyk2 construct, termed calcium regulated non kinase (CRNK) or green fluorescent protein (control) were expressed in GMC using adenovirus‐mediated gene transfer. ET‐1 stimulation resulted in a significant increase of Pyk2 phosphorylation accompanied by GTP‐loading of Rap1 and RhoA. CRNK expression inhibited ET‐1‐induced autophosphorylation of endogenous Pyk2 and diminished Rap1, but not RhoA, activation. The mechanism linking Pyk2 and Rap1 included (1) increased autophosphorylation of Pyk2 associated with p130Cas, (2) augmented p130Cas Y165 and Y249 phosphorylation, and (3) enhanced p130Cas–BCAR3 complex formation. CRNK expression prevented p130Cas phosphorylation and attenuated p130Cas association with BCAR3. Downregulation of endogenous BCAR3 protein expression using an siRNA technique led to a significant decrease in Rap1 activation in response to ET‐1. We observed that endogenous Pyk2 was important for GMC adhesion and spreading. Our data suggest that ET‐1 stimulated the GTPase Rap1 (but neither RhoA nor Ras) by a mechanism involving Pyk2 activation and recruitment of the p130Cas/BCAR3 complex in GMC. J. Cell. Physiol. 219: 45–56, 2009.

Targeting 20-HETE producing enzymes in cancer – rationale, pharmacology, and clinical potential

Studies demonstrate that lipid mediator 20-Hydroxyeicosatetraenoic acid (20-HETE) synthesis and signaling are associated with the growth of cancer cells in vitro and in vivo. Stable 20-HETE agonists promote the proliferation of cancer cells, whereas selective inhibitors of the 20-HETE-producing enzymes of the Cytochrome (CYP450)4A and CYP4F families can block the proliferation of glioblastoma, prostate, renal cell carcinoma, and breast cancer cell lines. A recent observation that the expression of CYP4A/4F genes was markedly elevated in thyroid, breast, colon, and ovarian cancer further highlights the significance of 20-HETE-producing enzymes in the progression of different types of human cancer. These findings provide the rationale for targeting 20-HETE-producing enzymes in human cancers and set the basis for the development of novel therapeutic strategies for anticancer treatment.

Cyclooxygenase 2: protein-protein interactions and posttranslational modifications

Numerous studies implicate the cyclooxygenase 2 (COX2) enzyme and COX2-derived prostanoids in various human diseases, and thus, much effort has been made to uncover the regulatory mechanisms of this enzyme. COX2 has been shown to be regulated at both the transcriptional and posttranscriptional levels, leading to the development of nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX2 inhibitors (COXIBs), which inhibit the COX2 enzyme through direct targeting. Recently, evidence of posttranslational regulation of COX2 enzymatic activity by s-nitrosylation, glycosylation, and phosphorylation has also been presented. Additionally, posttranslational regulators that actively downregulate COX2 expression by facilitating increased proteasome degradation of this enzyme have also been reported. Moreover, recent data identified proteins, located in close proximity to COX2 enzyme, that serve as posttranslational modulators of COX2 function, upregulating its enzymatic activity. While the precise mechanisms of the protein-protein interaction between COX2 and these regulatory proteins still need to be addressed, it is likely these interactions could regulate COX2 activity either as a result of conformational changes of the enzyme or by impacting subcellular localization of COX2 and thus affecting its interactions with regulatory proteins, which further modulate its activity. It is possible that posttranslational regulation of COX2 enzyme by such proteins could contribute to manifestation of different diseases. The uncovering of posttranslational regulation of COX2 enzyme will promote the development of more efficient therapeutic strategies of indirectly targeting the COX2 enzyme, as well as provide the basis for the generation of novel diagnostic tools as biomarkers of disease.

Pyk2 controls filamentous actin formation in human glomerular mesangial cells via modulation of profilin expression

Correspondence: Andrey Sorokin Medical College of Wisconsin, Department of Medicine/Kidney Disease Center, 8701 Watertown Plank Rd, Milwaukee, WI 53226, USA Tel +1 414 456 4438 Fax +1 414 456 6515 Email sorokin@mcw.edu Abstract: In glomerular mesangial cell (GMC), important regulators of glomerular filtration, adenovirus-mediated overexpression of calcium regulated nonkinase (CRNK), a dominant interfering calcium-regulated nonreceptor proline-rich tyrosine kinase 2 (Pyk2) construct, inhibited Pyk2 activity and caused enhanced RhoA activity, enriched cortical actin formation at time of cell replating, and reduction of spreading. We aimed to further explore Pyk2 regulation of the actin dynamic during cell spreading as a vital characteristic of GMC function. GMC were infected with adenovirus encoding CRNK or green fluorescent protein (GFP) as a control and 48 hours after infection cells were harvested and either re-plated or left in suspension for one hour. De novo adhesion to substrate was significantly decreased after Pyk2 activity inhibition and was further diminished after treatment with Rho-associated kinase inhibitor. Inhibition of Pyk2 was associated with increased filamentous actin formation and a corresponding decrease in globular to filamentous actin ratio during cell spreading. Phosphorylation and expression of cofilin, a RhoA-regulated filamentous actin destabilizing factor, were similar in CRNK-expressing and control GMC. Expression of profilin, an activator of actin polymerization, was enhanced, whereas phosphorylation of Pyk2 and p130Cas was decreased. Our data suggest that Pyk2 signaling controls the filamentous actin formation during cell spreading via upregulation of profilin expression.