Autophagy inhibition by chloroquine prevents increase in blood pressure and preserves endothelial functions

Purpose: To determine the effects of lysosomal inhibition of autophagy by chloroquine (CHQ) onhypertension-associated changes in the endothelial functions. Method: Angiotensin II (Ang II)-treated human endothelial cell line EA.hy926 and renovascularhypertensive rats were subjected to CHQ treatment (in vitro: 0.5, 1, and 2.5 μM; in vivo: 50 mg/kg/dayfor three weeks). Changes in the protein expressions of LC3b II (autophagosome formation marker) andp62 (autophagy flux marker) were assessed using immunoblotting. Cell migration assay, tubuleformation assay (in vitro), and organ bath studies (in vivo) were performed to evaluate the endothelialfunctions. Hemodynamic parameters were measured as well. Results: A higher expression of LC3b II and a reduced expression of p62 observed in the Ang II-treatedendothelial cells, as well as in the aorta of the hypertensive rats, indicated enhanced autophagy.Treatment with CHQ resulted in reduced autophagy flux (in vitro as well as in vivo) and suppressed AngII-induced endothelial cell migration and angiogenesis (in vitro). The treatment with CHQ was alsoobserved to prevent increase in blood pressure in hypertensive rats and preserved acetylcholineinducedrelaxation in phenylephrine-contracted aorta from the hypertensive rats. In addition, chloroquineattenuated Ang II-induced contractions in the aorta of normotensive as well as hypertensive rats. Conclusion: These observations indicated that CHQ lowers the blood pressure and preserves thevascular endothelial function during hypertension. Keywords: Angiotensin II, Autophagy, Chloroquine, Endothelial function, Hypertension, Vasculardysfunctio

Modulation of Insulin Resistance, Dyslipidemia and Serum Metabolome in iNOS Knockout Mice following Treatment with Nitrite, Metformin, Pioglitazone, and a Combination of Ampicillin and Neomycin

Oxidative and nitrosative stress plays a pivotal role in the incidence of metabolic disorders. Studies from this lab and others in iNOS-/- mice have demonstrated occurrence of insulin resistance (IR), hyperglycemia and dyslipidemia highlighting the importance of optimal redox balance. The present study evaluates role of nitrite, L-arginine, antidiabetics (metformin, pioglitazone) and antibiotics (ampicillin-neomycin combination, metronidazole) on metabolic perturbations observed in iNOS-/- mice. The animals were monitored for glucose tolerance (IPGTT), IR (insulin, HOMA-IR, QUICKI), circulating lipids and serum metabolomics (LC-MS). Hyperglycemia, hyperinsulinemia and IR were rescued by nitrite, antidiabetics, and antibiotics treatments in iNOS-/- mice. Glucose intolerance was improved with nitrite, metformin and pioglitazone treatment, while ampicillin-neomycin combination normalised the glucose utilization in iNOS-/- mice. Increased serum phosphatidylethanolamine lipids in iNOS-/- mice were reversed by metformin, pioglitazone and ampicillin-neomycin; dyslipidemia was however marginally improved by nitrite treatment. The metabolic improvements were associated with changes in selected serum metabolites-purines, ceramide, 10-hydroxydecanoate, glucosaminate, diosmetin, sebacic acid, 3-nitrotyrosine and cysteamine. Bacterial metabolites-hippurate, indole-3-ethanol; IR marker-aminoadipate and oxidative stress marker-ophthalmate were reduced by pioglitazone and ampicillin-neomycin, but not by nitrite and metformin treatment. Results obtained in the present study suggest a crucial role of gut microbiota in the metabolic perturbations observed in iNOS-/- mice

Ameliorating impaired cardiac function in myocardial infarction using exosome-loaded gallic-acid-containing polyurethane scaffolds

Myocardial infarction (MI) can be tackled by implanting cardiac patches which provide mechanical support to the heart. However, most tissue-engineered scaffolds face difficulty in attenuating oxidative stress, maintaining mechanical stability, and regenerating damaged cardiomyocytes. Here, we fabricated elastic cryogels using polyurethane modified with antioxidant gallic acid in its backbone (PUGA) and further coated them with decellularized extracellular matrix (dECM) to improve adhesiveness, biocompatibility and hemocompatibility. The scaffold was functionalized with exosomes (EXO) isolated from adipose-derived stem cells having regenerative potential. PUGA-dECM + EXO was tested in a rat model with induced MI where echocardiography after 8 weeks of implantation showed significant recovery in treatment group. Histological analysis revealed a decrease in fibrosis after application of patch and promotion of angiogenesis with reduced oxidative stress was shown by immunostaining. Expression of cardiac tissue contractile function marker was also observed in treatment groups. Thus, the proposed biomaterial has a promising application to be utilized as a patch for cardiac regeneration. More detailed studies with larger animal species are needed for using these observations for specific applications

FGF21 promotes endothelial cell angiogenesis through a dynamin-2 and Rab5 dependent pathway.

Binding of angiogenic molecules with cognate receptor tyrosine kinases (RTK) is required for angiogenesis however the precise link between RTK binding, endocytosis, and signaling requires further investigation. Here, we use FGFR1 as a model to test the effects of the large GTPase and endocytosis regulatory molecule dynamin-2 on angiogenic signaling in context of distinct FGF ligands. In vitro, overexpression of dominant negative dynamin-2 (DynK44A) attenuates FGFR1 activation of Erk and tubulogenesis by FGF2. Furthermore, we identify FGF21, a non-classical, FGF ligand implicated in diverse human pathologies as an angiogenic molecule acting through FGFR1 and β-Klotho coreceptor. Disruption of FGFR1 activation of ERK by FGF21 is achieved by perturbation of the function of both dynamin-2 and Rab5 GTPase. In vivo, mice harboring endothelial selective overexpression of DynK44A, show impaired angiogenesis in response to FGF21. In conclusion, dynamin dependent endocytosis of FGFR1 is required for in vitro and in vivo angiogenesis in response to FGF2 and the non-classical FGF ligand, FGF21. These studies extend our understanding of the relationships between RTK binding, internalization, endosomal targeting, and angiogenic signaling

Systemic deficiency of the MAP kinase-activated protein kinase 2 reduces atherosclerosis in hypercholesterolemic mice

Atherosclerosis is a chronic inflammatory disease and represents the major cause of cardiovascular morbidity and mortality. A critical regulator of inflammatory processes represents the mitogen-activated protein kinase-activated protein kinase-2 (MK2). Therefore, we investigated the functional role of MK2 in atherogenesis in hypercholesterolemic mice as well as potentially underlying mechanisms in vivo and in vitro. Activation of MK2 (phospho-MK2) was predominantly detected in the endothelium and macrophage-rich plaque areas within aortas of hypercholesterolemic LDL receptor-deficient mice (ldlr(-/-)). Systemic MK2 deficiency of hypercholesterolemic ldlr(-/-) mice ( ldlr(-/-)/mk2(-/-)) significantly decreased the accumulation of lipids and macrophages in the aorta after feeding an atherogenic diet for 8 and 16 weeks despite a significant increase in proatherogenic plasma lipoproteins compared with ldlr(-/-) mice. Deficiency of MK2 significantly decreased oxLDL-induced foam cell formation in vitro, diet-induced foam cell formation in vivo, and expression of scavenger receptor A in primary macrophages. In addition, systemic MK2 deficiency of hypercholesterolemic ldlr(-/-) mice significantly decreased the aortic expression of the adhesion molecule VCAM-1 and the chemokine MCP-1, key mediators of macrophage recruitment into the vessel wall. Furthermore, silencing of MK2 in endothelial cells by siRNA reduced the IL-1 beta-induced expression of VCAM-1 and MCP-1. MK2 critically promotes atherogenesis by fostering foam cell formation and recruitment of monocytes/macrophages into the vessel wall. Therefore, MK2 might represent an attractive novel target for the treatment of atherosclerotic cardiovascular disease

FGF21 promotes angiogenesis <i>in vitro</i> and <i>in vivo</i>.

<p>A. EC were plated on growth factor reduced Matrigel and stimulated with FGF2 and tubulogenesis was measured after 6(n = 3, *p<0.05). B. EC with and without overexpression of β-Klotho were plated on Matrigel and treated with FGF21 and analyzed after 6 hours for tube formation (n = 3, *p<0.05). C. Confluent monolayer of EC with βKotho overexpression were scratched and treated with FGF21 and analyzed for cell migration by measuring width of scratch. (n = 3, *p<0.05). D and E. Directed <i>in vivo</i> angiogenesis assay was carried out in mice using growth factor reduced basement membrane extract with and without FGF2 (D) and FGF21 (E). The silicon tubes were implanted subcutaneously. After two weeks, mice were sacrificed and tubes recovered and analyzed for angiogenesis by imaging using Leica MZ125 microscope. Vascular invasion was quantified using MetaMorph Imaging software (n = 3, *p<0.05).</p

DynK44A blocks FGF21 mediated angiogenesis.

<p>A. Basement membrane extracts were premixed with AdCre or Ad LacZ adenovirus along with FGF21 and filled into the silicon tubes before subcutaneous implantation in DynK44A^fl/fl mice. 2 weeks after implantation, mice were sacrificed and tubes were recovered, and analyzed for hemoglobin quantitation. Images of basement membrane extract from directed <i>in vivo</i> angiogenesis, H&E stain from paraffin sections of extract recovered from mouse implants, and hemoglobin quantitation are all shown (*p<0.05). Arrows indicate vascularity. B. Basement membrane extracts were premixed with FGF21, filled into the silicon tubes and implanted in TIE2^cre/DynK44A^fl/fl or DynK44A^fl/fl mice. Vascular invasion was quantified using MetaMorph Imaging software images from directed <i>in vivo</i> angiogenesis are shown (n = 4, *p<0.05).</p