The vascular endothelial growth factor trap aflibercept induces vascular dysfunction and hypertension via attenuation of eNOS/NO signaling in mice.

Aflibercept, as a soluble decoy vascular endothelial growth factor receptor, Which has been used as a first-line monotherapy for cancers. Aflibercept often causes cardiovascular toxicities including hypertension, but the mechanisms underlying aflibercept-induced hypertension remain unknown. In this study we investigated the effect of short-term and long-term administration of aflibercept on blood pressure (BP), vascular function, NO bioavailability, oxidative stress and endothelin 1 (ET-1) in mice and cultured endothelial cells. We showed that injection of a single-dose of aflibercept (18.2, 36.4 mg/kg, iv) rapidly and dose-dependently elevated BP in mice. Aflibercept treatment markedly impaired endothelial-dependent relaxation (EDR) and resulted in NADPH oxidases 1 (NOX1)- and NADPH oxidases 4 (NOX4)-mediated generation of ROS, decreased the activation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) concurrently with a reduction in nitric oxide (NO) production and elevation of ET-1 levels in mouse aortas; these effects were greatly attenuated by supplementation of L-arginine (L-arg, 0.5 or 1.0 g/kg, bid, ig) before aflibercept injection. Similar results were observed in L-arg-pretreated cultured endothelial cells, showing markedly decreased ROS accumulation and AKT/eNOS/NO signaling impairment induced by aflibercept. In order to assess the effects of long-term aflibercept on hypertension and to evaluate the beneficial effects of L-arg supplementation, we administered these two drugs to WT mice for up to 14 days (at an interval of two days). Long-term administration of aflibercept resulted in a sustained increase in BP and a severely impaired EDR, which are associated with NOX1/NOX4-mediated production of ROS, increase in ET-1, inhibition of AKT/eNOS/NO signaling and a decreased expression of cationic amino acid transporter (CAT-1). The effects caused by long-term administration were greatly attenuated by L-arg supplementation in a dose-dependent manner. We conclude that aflibercept leads to vascular dysfunction and hypertension by inhibiting CAT-1/AKT/eNOS/NO signaling, increasing ET-1, and activating NOX1/NOX4-mediated oxidative stress, which can be suppressed by supplementation of L-arg. Therefore, L-arg could be a potential therapeutic agent for aflibercept-induced hypertension.

Genetic ablation and pharmacological inhibition of immunosubunit ß5i attenuates cardiac remodeling in deoxycorticosterone-acetate (DOCA)-salt hypertensive mice.

Hypertensive cardiac remodeling is a major cause of heart failure. The immunoproteasome is an inducible form of the proteasome and its catalytic subunit ß5i (also named LMP7) is involved in angiotensin II-induced atrial fibrillation; however, its role in deoxycorticosterone-acetate (DOCA)-salt-induced cardiac remodeling remains unclear. C57BL/6 J wild-type (WT) and ß5i knockout (ß5i KO) mice were subjected to uninephrectomy (sham) and DOCA-salt treatment for three weeks. Cardiac function, fibrosis, and inflammation were evaluated by echocardiography and histological analysis. Protein and gene expression levels were analyzed by quantitative real-time PCR and immunoblotting. Our results showed that after 21 days of DOCA-salt treatment, ß5i expression and chymotrypsin-like activity were the most significantly increased factors in the heart compared with the sham control. Moreover, DOCA-salt-induced elevation of blood pressure, adverse cardiac function, chamber and myocyte hypertrophy, interstitial fibrosis, oxidative stress, and inflammation were markedly attenuated in ß5i KO mice. These findings were verified in ß5i inhibitor PR-957-treated mice. Moreover, blocking of PTEN (the gene of phosphate and tensin homolog deleted on chromosome ten) markedly attenuated the inhibitory effect of ß5i knockout on DOCA-salt-induced cardiac remodeling. Mechanistically, DOCA-salt stress upregulated the expression of ß5i, which promoted the degradation of PTEN and the activation of downstream signals (AKT/mTOR, TGF-ß1/Smad2/3, NOX, and NF-κB), which ultimately led to cardiac hypertrophic remodeling. This study provides new evidence of the critical role of ß5i in DOCA-salt-induced cardiac remodeling through the regulation of PTEN stability, and indicates that the inhibition of ß5i may be a promising therapeutic target for the treatment of hypertensive heart diseases.

Resveratrol Attenuates Pressure Overload-Induced Cardiac Fibrosis and Diastolic Dysfunction via PTEN/AKT/Smad2/3 and NF-κB Signaling Pathways.

SCOPE: Cardiac fibrosis is a key feature of cardiac remodeling. Recently, a protective role for resveratrol (RES) in pressure-overload-induced cardiac hypertrophy and contractile dysfunction has been demonstrated. However, the effect of RES on cardiac fibrosis and diastolic function in this model remains unclear. METHODS AND RESULTS: Cardiac remodeling is induced in mice by transverse aortic constriction (TAC) for 2-4 weeks. RES is administered at dose of 5 or 50 mg kg-1  d-1 for 2 weeks. It is found that RES administration at 50 mg kg-1  d-1 significantly attenuates TAC-induced adverse cardiac systolic and diastolic function, fibrosis, inflammation, and oxidative stress via inhibiting PTEN degradation and the downstream mediators. However, RES at 5 mg kg-1  d-1 has no significant effects. RES at 50 mg kg-1  d-1 also ameliorates pre-established adverse cardiac function and remodeling induced by TAC. Treatment with PTEN inhibitor VO-OHpic (10 mg kg-1  d-1 ) for 2 weeks abolishes RES-mediated protective effects. Additionally, the effect of RES (100 µm) on inhibition of Ang II-induced fibroblast proliferation and activation in vitro is verified. CONCLUSIONS: The findings provide new evidence that RES plays a critical role in the progression of cardiac fibrosis and diastolic dysfunction, and suggest that RES may be a promising therapeutic agent for cardiac fibrosis.

Chronic inhibition of chemokine receptor CXCR2 attenuates cardiac remodeling and dysfunction in spontaneously hypertensive rats.

System hypertension is a major risk factor for cardiac hypertrophy and heart failure. Our recent findings reveal that the ablation or inhibition of C-X-C chemokine receptor (CXCR) 2 blocks this process in mice; however, it is not clear whether the pharmacological inhibition of CXCR2 attenuates hypertension and subsequent cardiac remodeling in spontaneously hypertensive rats (SHRs). In the present study, we showed that chemokines (CXCL1 and CXCL2) and CXCR2 were significantly upregulated in SHR hearts compared with Wistar-Kyoto rat (WKY) hearts. Moreover, the administration of CXCR2-specific inhibitor N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)-urea (SB225002) in SHRs (at 2 months of age) for an additional 4 months significantly suppressed the elevation of blood pressure, cardiac myocyte hypertrophy, fibrosis, inflammation, and superoxide production and improved heart dysfunction in SHRs compared with vehicle-treated SHRs. SB225002 treatment also reduced established hypertension, cardiac remodeling and contractile dysfunction. Moreover, CXCR2-mediated increases in the recruitment of Mac-2-positive macrophages, proinflammatory cytokines, vascular permeability and ROS production in SHR hearts were markedly attenuated by SB225002. Accordingly, the inhibition of CXCR2 by SB225002 deactivates multiple signaling pathways (AKT/mTOR, ERK1/2, STAT3, calcineurin A, TGF-ß/Smad2/3, NF-κB-p65, and NOX). Our results provide new evidence that the chronic blocking of CXCR2 activation attenuates progression of cardiac hypertrophic remodeling and dysfunction in SHRs. These findings may be of value in understanding the benefits of CXCR2 inhibition for hypertensive cardiac hypertrophy and provide further support for the clinical application of CXCR2 inhibitors for the prevention and treatment of heart failure.

Time Series Gene Expression Profiling and Temporal Regulatory Pathway Analysis of Angiotensin II Induced Atrial Fibrillation in Mice.

Background/Aim: Angiotensin II (Ang II) and hypertension play critical roles in the pathogenesis of the atrial remodeling that contributes to atrial fibrillation (AF). However, the gene expression profiles and signaling pathways in atria during the development of AF induced by Ang II remain unknown. Methods: Wild-type male mice (C57BL/6 background, 10 weeks old) were administered an infusion of Ang II (2000 ng/kg/min) using an osmotic pump for 1, 2, and 3 weeks. Blood pressure (BP) was measured by the tail-cuff method. AF was induced and recorded. Atrial enlargement and remodeling were examined by echocardiography and Masson's trichrome staining. Time-series microarray analyses were conducted to examine gene expression profiles and pathways. Results: Ang II infusion resulted in marked elevation of systolic BP, increased AF incidence and duration, atrial enlargement, fibrosis, and atrial infiltration of myofibroblasts and F4/80-positive macrophages in a time-dependent manner. Microarray results showed that 1,719 genes were differentially expressed in the atrium at weeks 1, 2, and 3 after Ang II infusion. Gene ontology showed that these genes participate mainly in immune system processes, and regulation of cell migration, cell adhesion, complement activation, and the inflammatory response. Significant pathways included lysosomal and phagosomal pathways, which are involved in antigen processing and presentation, as well as chemokine signaling, and extracellular matrix-receptor interaction, which are known to play important roles in Ang II-induced AF. Moreover, these differentially expressed genes were classified into 50 profiles by hierarchical cluster analysis. Of these, eight profiles were significant and contained a total of 1,157 genes. Gene co-expression network analysis identified that Pik3cg (also known as phosphoinositide-3-kinase regulatory subunit 3) was localized in the core of the gene network, and was the most highly expressed among the Pik3 isoforms at different time points. Conclusion: The present findings revealed that many genes are involved in Ang II-induced AF, and highlighted that Pik3cg may play a central role in this disease.

Resveratrol as a new inhibitor of immunoproteasome prevents PTEN degradation and attenuates cardiac hypertrophy after pressure overload.

Sustained cardiac hypertrophy is a major cause of heart failure (HF) and death. Recent studies have demonstrated that resveratrol (RES) exerts a protective role in hypertrophic diseases. However, the molecular mechanisms involved are not fully elucidated. In this study, cardiac hypertrophic remodeling in mice were established by pressure overload induced by transverse aortic constriction (TAC). Cardiac function was evaluated by echocardiography and invasive pressure-volume analysis. Cardiomyocyte size was detected by wheat germ agglutinin staining. The protein and gene expressions of signaling mediators and hypertrophic markers were examined. Our results showed that administration of RES significantly suppressed pressure overload-induced cardiac hypertrophy, fibrosis and apoptosis and improved in vivo heart function in mice. RES also reversed pre-established hypertrophy and restoring contractile dysfunction induced by chronic pressure overload. Moreover, RES treatment blocked TAC-induced increase of immunoproteasome activity and catalytic subunit expression (ß1i, ß2i and ß5i), which inhibited PTEN degradation thereby leading to inactivation of AKT/mTOR and activation of AMPK signals. Further, blocking PTEN by the specific inhibitor VO-Ohpic significantly attenuated RES inhibitory effect on cardiomyocyte hypertrophy in vivo and in vitro. Taken together, our data suggest that RES is a novel inhibitor of immunoproteasome activity, and may represent a promising therapeutic agent for the treatment of hypertrophic diseases.

Gallic Acid Suppresses Cardiac Hypertrophic Remodeling and Heart Failure.

SCOPE: Gallic acid (GA) is a dietary phenolic acid found in tea, red wine, and some plants. It exhibits anti-oxidative and anti-inflammatory activities. Recent studies have revealed that GA has beneficial effects against several cardiovascular diseases; however, whether GA attenuates pressure-overload-induced cardiac hypertrophy and the underlying mechanism remains unclear. METHODS AND RESULTS: Primary cardiomyocyte hypertrophy is stimulated with angiotensin II (Ang II). Cardiac hypertrophic remodeling is induced in mice by transverse aortic constriction (TAC). Myocardial function is evaluated by echocardiographic and hemodynamic analyses, while cardiac tissues are analyzed by histological staining. It is observed that GA significantly decreases Ang II-induced increases in cardiomyocyte size in vitro. Administration of GA in mice markedly improves TAC-induced cardiac dysfunction and attenuates pathological changes, including cardiac myocyte hypertrophy, fibrosis, inflammation, and oxidative stress. Mechanistically, GA inhibits ULK1 and activates autophagy, which induces the degradation of EGFR, gp130, and calcineurin A, thereby inhibiting the downstream signaling cascades (AKT, ERK1/2, JAK2/STAT3, and NFATc1). CONCLUSIONS: The results demonstrate for the first time that GA prevents myocardial hypertrophy and dysfunction via an autophagy-dependent mechanism. Thus, GA represents a promising therapeutic candidate for treating cardiac hypertrophy and heart failure.

Analysis of Genes Related to Angiotensin II-Induced Arterial Injury Using a Time Series Microarray.

BACKGROUND/AIMS: Angiotensin II (Ang II)-mediated hypertension is a major risk factor for cardiovascular diseases. Ang II induces changes in vessel structure and function through the activation of genes related to signaling pathways. However, the changes in the gene expression profiles of blood vessels in response to Ang II remain unclear. METHODS: Wild-type C57BL/6 mice were infused with Ang II (1500 ng/kg/min) using an osmotic pump for 1, 3, and 7 days. Vascular wall inflammation and remodeling were evaluated by pathological examination. Time-series microarray and quantitative PCR analyses were performed. Bioinformatics analyses were conducted to identify key genes, pathways, and biological processes. RESULTS: After Ang II infusion, blood pressure and aortic remodeling were increased over time. Microarray analysis identified a totally of 3631 differentially expressed genes in aortas at days 1, 3, and 7 of Ang II infusion. These genes were involved in multiple biological processes, including cell adhesion, angiogenesis, cell migration, protein phosphorylation, immune system, and cell cycle, which may play important roles in regulating Ang II-induced arterial injury during hypertension. The genes were classified into 50 profiles by hierarchical cluster analysis, and finally, 14 significant profiles were identified. Among these genes, protein kinase cAMP-activated catalytic subunit alpha (Prkaca), a gene that directly regulated 137 neighboring genes, was located at the center of the gene network in Ang II-infused aortas. Further, Prkaca protein expression and cAMP level were downregulated in a time-dependent manner in Ang II-infused aortas. CONCLUSIONS: The combined use of DNA microarrays and cluster and gene network analyses identified Prkaca as a key Ang II-responsive gene that may mediate early vascular injury and hypertension.