Zhang, Heyu

Background: Cardiac fibrosis is a central pathological process that leads to worse outcomes in various cardiomyopathies. Recent evidence shows that CDCP1 (CUB domain-containing protein 1) modulates myocardial recovery by attenuating human cardiac fibroblast (HCF) proliferation. This study investigates the hypothesis that selective targeting of the CDCP1 cleavage domain can mitigate fibroblast proliferation, thereby offering a novel therapeutic approach to reducing cardiac fibrosis. Methods: A high-throughput in silico screening was performed using the Enamine compound library, focusing on structural regions that include the cleavage and adjacent sites of CDCP1 . A total of 147 candidate compounds were prioritized based on docking scores and binding stability profiles. Selected compounds were subsequently evaluated in vitro using HCF cultures seeded at 3,000 cells/well in 96-well plates. Each compound was administered at 10 μM concentration, and cell proliferation and viability were assessed by time-lapse imaging in the Incucyte® system at 24 and 96 hours. All experiments were done in triplicate with appropriate DMSO vehicle controls. Results: Compound C3 significantly reduced HCF proliferation (Fig B) to 27.3±2.4% relative to DMSO controls (99.9±0.5%, P <0.001) at 96 hours. Detailed structural modeling indicated that C3 binds to the CDCP1 cleavage site (Fig C) with a favorable Glide XP docking score of -7.66 kcal/mol. The interaction is stabilized by hydrogen bonds with Lys365 and Cys374 (Fig D), and supplementary hydrophobic interactions between aromatic rings and aliphatic groups, which contribute to a strong inhibitory effect. Conclusion: Targeting the CDCP1 cleavage domain with compound C3 effectively diminishes HCF proliferation in vitro. Therefore, these results substantiate the therapeutic potential of CDCP1 inhibition in cardiac fibrosis. Ongoing in vivo studies will explore the translational relevance of these findings.

Activation of soluble guanylate cyclase (sGC), a key downstream target of nitric oxide (NO) signaling, is reduced in valve tissue from patients with fibrocalcific aortic valve stenosis (FCAVS). We hypothesized that restoring NO signaling pathway using Ataciguat (ATA)—an activator of oxidized sGC—would slow progression of aortic valve calcification. To determine whether Ataciguat attenuates bone morphogenetic protein (BMP2) signaling in vitro, we treated mouse aortic valve interstitial cells with exogenous BMP2(200ng/ml) with or without ATA(10nm), and demonstrated that ATA significantly reduced canonical BMP2 signaling by more than 50% (p< 0.01). To determine whether Ataciguat slows progression of FCAVS in vivo , ldlr -/- /apoB 100/100 mice were assigned to one of two groups:1) 9 months of western diet feeding (WD), or 2) 6 months of WD followed by 3 months of WD + ATA (5mg/kg ATA). In this model, Ataciguat attenuated BMP signaling in aortic valve tissue (p < 0.05), reduced aortic valve calcification (p < 0.05), and prevented progression of aortic valve dysfunction (p < 0.05). Finally, to determine whether ATA slows progression of calcification in humans, we treated patients with mild-to-moderate FCAVS with placebo or ATA (200 mg/day) for 6 months. In this Phase II clinical trial, ATA significantly slowed progression of aortic valve calcification (computed tomography imaging; p = 0.05) and also tended to slow progressive reductions in aortic valve area and valvular stenosis. Collectively, we believe this is the first study to demonstrate the pre-clinical and clinical utility of pharmacologically stimulating sGC to attenuate osteogenic signaling and slow progression of aortic valve disease in mice and humans, and creates a unique opportunity for larger trials focused on the effectiveness of this strategy to delay or prevent surgery in FCAVS.

While the strong connection between oxidative stress and atherosclerosis has many to hypothesize that antioxidant therapy may be viable therapeutic target in combating progression of atherosclerosis, numerous clinical trials of antioxidant interventions that scavenge multiple reactive oxygen species have been negative. Here, we test the hypothesis that overexpression of catalase—an enzyme which selectively degrades H 2 O 2 —plays a protective role against the initiation and expansion of calcific lesions in advanced atherosclerotic plaques. In vitro, we used aortic smooth muscle cells (SMC) from Ldlr -/- ApoB 100/100 (LA) mice that were either wild-type for human catalase (LA-Cat 0/0 ) or overexpressing human catalase (LA-Cat Tg/0 ). After treatment with BMP2 or TNFα for 24 hours, we found that catalase-overexpressing VSMCs had modest reductions in TNFα-induced inflammatory markers but paradoxical increases in mRNA levels of BMP2-induced osterix and the SMAD-specific target ID1. To determine if these effects were conserved in vivo, we analyzed aortic tissue from LA mice fed a Western diet for 3 or 12 months. Surprisingly—and unlike our cell-based experiments—aorta from LA-Cat Tg/0 tended to have higher expression of inflammatory markers compared to their LA-Cat 0/0 littermates. Similarly, expression of osterix was increased in LA-Cat Tg/0 mice compare to their LA-Cat 0/0 littermates, which was also associated with an increase in intimal plaque calcification. Collectively, these data strongly suggest that systemically overexpression of catalase (a close parallel to systemic administration of a selective H 2 O 2 -scavenging antioxidant) does not result in molecular, histopathological, or functional benefits in large arteries from hypercholesterolemic mice. Future studies aimed at characterizing the complex interplay between reactive oxygen species and specific intracellular signaling pathways are an essential next step to understanding the role of endogenous reactive oxygen species in the pathogenesis of atherosclerotic vascular disease.

BACKGROUND: Pharmacological treatments for fibrocalcific aortic valve stenosis (FCAVS) have been elusive for >50 years. Here, we tested the hypothesis that reactivation of oxidized sGC (soluble guanylate cyclase), the primary receptor for nitric oxide, with ataciguat is a safe and efficacious strategy to slow progression of FCAVS. METHODS: We used quantitative real-time reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry to characterize sGC signaling and the biological effects of ataciguat on signaling cascades related to nitric oxide, calcification, and fibrosis in excised human aortic valve tissue, aortic valve interstitial cells, and mouse aortic valves. We then conducted randomized, placebo-controlled phase I (14-day safety/tolerance) and phase II (6-month efficacy) trials in patients with moderate aortic valve stenosis. RESULTS: In excised human tissue, we found robust losses in sGC signaling despite upregulation of sGC subunits. In vitro, ataciguat increased sGC signaling and reduced BMP2 (bone morphogenetic protein 2) signaling in aortic valve interstitial cells. In mice with established FCAVS, treatment with ataciguat attenuated BMP signaling and slowed progression of valve calcification and dysfunction. In a phase I, randomized, placebo-controlled trial, treatment with ataciguat for 2 weeks was safe and well tolerated in patients with moderate FCAVS ( https://www.clinicaltrials.gov ; Unique identifier: NCT02049203). In a separate phase II, randomized, placebo-controlled trial, treatment with ataciguat for 6 months slowed the progression of aortic valve calcification and tended to slow the progression of valvular and ventricular dysfunction in patients with moderate FCAVS ( https://www.clinicaltrials.gov ; Unique identifier: NCT02481258). CONCLUSIONS: Collectively, this study highlights the therapeutic potential of the targeted restoration of the diseased/inactive form of sGC for treatment of FCAVS. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02049203. URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02481258.

Increases in vascular calcification (VC) and fibrosis are a near ubiquitous finding with aging. Here, we sought to test the hypothesis that Metformin (MET)—an AMPK activator entering large clinical trials that is thought to attenuate multimorbidity associated with aging—attenuates key molecular processes known to drive vascular calcification (VC) and fibrogenic signaling in vascular smooth muscle cells (VSMCs) in vitro. Given recent reports highlighting the importance of mitochondria and sirtuins with aging, we also tested the hypothesis that MET-driven reductions in osteogenic signaling are associated with upregulation of SIRT3. We used exogenous BMP2 (100 ng/ml) and TGFβ1 (10 ng/ml) as osteogenic and fibrogenic stimuli, respectively. Consistent with prior work from our lab, treatment with BMP2 for 24 hours robustly increased expression of the osteogenic genes Msx2 (3.9±1.5 fold-change, FC), Runx2 (3.5±1.2 FC), and osterix (17.2±5.2 FC) in VSMCs. Critically, co-treatment with Metformin dramatically reduced expression of these genes by 68 ± 22%, 58 ±20%, and 63 ± 4%, respectively. Subsequent experiments treating cells with exogenous TGFβ1 resulted in significant induction of the fibrogenic marker/known TGFβ1 target versican (1.7±0.4 FC), an effect that was largely attenuated by co-treatment with MET (58±3%). Treatment with MET did not, however, impact TGFβ1-induced increases in CTGF or fibronectin. Treatment with either BMP2 or TGFβ1 resulted in significant increases in mRNA levels of SIRT3 (2.5 ±0.11 and 1.4 ±0.17 FC, respectively), whereas treatment with MET resulted in modest and less consistent increases in SIRT3 mRNA (1.3 ± 0.2 FC). Interestingly, however, co-treatment with BMP2+MET or TGFβ1+MET resulted in uniform suppression of BMP2/ TGFβ1-induced increases in SIRT3 (54±19% and 25±12%, respectively). Collectively, these data suggest that treatment with MET may be a viable strategy to attenuate age-associated increases in osteogenic and fibrogenic signaling in conduit vessels, and suggest further in vivo investigation into therapeutic efficacy is warranted.

Cardiovascular calcification is often an active process involving re-differentiation of cells to an osteoblast-like phenotype, and is strongly associated with aging. Sirtuins are histone and protein deacetylases that play a critical role in a number of age-related diseases, and SIRT6-deficient mice have the most profound progeroid phenotype. The role of SIRT6 in the regulation of cardiovascular calcification, however, is poorly understood. We hypothesized that age-related reductions in sirtuins promote osteogenic differentiation in cells from aortic valve and aorta. We used aortic valve interstitial cells isolated from SIRT6-deficient mice, pigs, or humans, and vascular smooth muscle cells from mouse aorta to determine whether osteogenic differentiation of cardiovascular cells is regulated by SIRT6 levels. In line with our hypothesis, we found increased p-SMAD1/5/8 levels in cells from SIRT6-deficient aortic valve and aorta. Second, global SIRT inhibition with NAD+-depletion increased histone acetylation and protein levels of the osteogenic proteins Sp7 and Runx2. Third, inhibition of SIRTs enhanced the transcriptional responses of Sp7 and Runx2 when cells were treated with BMP2. Fourth, knockdown of SIRT6 in mouse and human aorta and aortic valve cells by RNA interference increased Sp7 expression. Finally, chronic pharmacological inhibition of SIRTs promoted calcified nodule formation and calcium deposition in cultured mouse/swine aortic valve cells. Collectively, our data suggest that reductions in SIRT6 levels favor amplification of pro-osteogenic signaling and osteogenic differentiation of cardiovascular cells and promote the formation of calcified nodules in vitro . These data also suggest that sirtuin re-activation may be a useful therapy to slow initiation and/or progression of cardiovascular calcification in vivo.

Increasing age is a major risk factor for calcific aortic valve disease (CAVD). Interestingly, SIRT6 knockout mice have a marked progeroid phenotype, and we recently reported that sirtuin enzyme expression is dramatically reduced with aging and that SIRT6 expression is reduced further in valves from patients with end-stage CAVD. It is unknown, however, whether experimentally reducing SIRT6 promotes osteogenic signaling in the valve and ultimately accelerates progression of CAVD. Thus, we used cultured mouse aortic valve interstitial cells and ldlr-deficient, apolipoprotein B100-only mice (LA) that were SIRT6 wild-type (LA-SIRT6 +/+ ) or heterozygous (LA-SIRT6 +/- ) and fed a Western diet for 3 or 12 months to determine the role of SIRT6 in valve calcification. In vitro , reduction of SIRT6 increased histone acetylation and significantly increased mRNA and protein levels of the osteogenic genes Runx2 and Sp7 in response to bone morphogenetic protein 2 (100ng/ml BMP2 for 18 hours), and siRNA knockdown of SIRT6 increased mRNA levels of Sp7 even in the absence of exogenous bone morphogens. Using high-resolution ultrasound to evaluate aortic valve function in vivo, we found that 3 month old LA-SIRT6 +/- mice did not have significant impairments in valve function compared to LA-SIRT6 +/+ mice. In contrast, 12 month old LA-SIRT6 +/- had dramatically worsened aortic valve dysfunction and stenosis compared to LA-SIRT6 +/+ mice, which was also associated with reductions in left ventricular ejection fraction. Collectively, our data strongly suggest SIRT6 plays a critical role in the tonic repression of osteogenic signaling in the aortic valve, and that age-related reductions in SIRT6 are likely to increase susceptibility to valve calcification in response to risk factors for CAVD such as hypercholesterolemia. Collectively, increasing activity of SIRT6 or reducing acetylation of its targets may serve as viable therapeutic strategies to slow progression of age-related valvular calcification and stenosis.

Abstract Neuropilin-2 (NRP2) is a non-tyrosine kinase receptor frequently overexpressed in various malignancies, where it has been implicated in promoting many protumorigenic behaviors, such as imparting therapeutic resistance to metastatic cancer cells. Here, we report a novel function of NRP2 as a regulator of endocytosis, which is enhanced in cancer cells and is often associated with increased metastatic potential and drug resistance. We found that NRP2 depletion in human prostate and pancreatic cancer cells resulted in the accumulation of EEA1/Rab5-positive early endosomes concomitant with a decrease in Rab7-positive late endosomes, suggesting a delay in early-to-late endosome maturation. NRP2 depletion also impaired the endocytic transport of cell surface EGFR, arresting functionally active EGFR in endocytic vesicles that consequently led to aberrant ERK activation and cell death. Mechanistic investigations revealed that WD-repeat– and FYVE-domain–containing protein 1 (WDFY1) functioned downstream of NRP2 to promote endosome maturation, thereby influencing the endosomal trafficking of EGFR and the formation of autolysosomes responsible for the degradation of internalized cargo. Overall, our results indicate that the NRP2/WDFY1 axis is required for maintaining endocytic activity in cancer cells, which supports their oncogenic activities and confers drug resistance. Therefore, therapeutically targeting endocytosis may represent an attractive strategy to selectively target cancer cells in multiple malignancies. Cancer Res; 76(2); 418–28. ©2015 AACR.

Background— Pathological processes underlying myxomatous mitral valve degeneration (MMVD) remain poorly understood. We sought to identify novel mechanisms contributing to the development of this condition. Methods and Results— Microarrays were used to measure gene expression in 11 myxomatous and 11 nonmyxomatous human mitral valves. Differential gene expression (thresholds P <0.05; fold-change >1.5) and pathway activation (Ingenuity) were confirmed using quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Contributions of bone morphogenetic protein 4 and transforming growth factor (TGF)-β2 to differential gene expression were evaluated in vitro. Contributions of angiotensin II to differential pathway activation were examined in mice in vivo. A total of 2602 genes were differentially expressed between myxomatous and nonmyxomatous valves. Canonical TGF-β signaling was increased in MMVD because of increased ligand expression and derepression of SMA mothers against decapentaplegic 2/3 signaling and was confirmed with quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Myxomatous valves demonstrated activation of canonical bone morphogenetic protein and Wnt/β-catenin signaling and upregulation of their common target runt-related transcription factor 2. Our data set provided transcriptional and immunohistochemical evidence for activated immune cell infiltration. In vitro treatment of mitral valve interstitial cells with TGF-β2 increased β-catenin signaling at mRNA and protein levels, suggesting interactions between TGF-β2 and Wnt signaling. In vivo infusion of mice with angiotensin II recaptured several changes in signaling pathways characteristic of human MMVD. Conclusions— These data support a new disease framework whereby activation of TGF-β2, bone morphogenetic protein 4, Wnt/β-catenin, or immune signaling plays major roles in the pathogenesis of MMVD. We propose these pathways act in a context-dependent manner to drive phenotypic changes that fundamentally differ from those observed in aortic valve disease and open novel avenues guiding future research into the pathogenesis of MMVD.

Abstract A major contributor to cancer mortality is recurrence and subsequent metastatic transformation following therapeutic intervention. Therefore, in order to develop new treatment modalities and improve the efficacy of current ones, it is important to understand the molecular mechanisms that promote resistance to therapy in cancer cells. One pathway contributing to therapy resistance is autophagy, a self-digestive process that can eliminate unnecessary or damaged organelles to protect cancer cells from death. We have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which helps cancer cell survival following treatment. Inhibition of mTOR complex 1 activity by this axis is the underlying mechanism for the activation of autophagy. Furthermore, we identified two VEGF-C/NRP-2-regulated genes, LAMP-2 and WDFY-1, that have previously been suggested to participate in autophagy and vesicular trafficking. Upregulation of WDFY-1 following VEGF-C or NRP-2 depletion contributes to cytotoxic drug-mediated cell death. Together, these data suggest a link between the VEGF-C/NRP-2 axis and cancer cell survival despite the presence of chemotherapy-induced stress. Effective targeting of this pathway may lead to the development of new cancer therapies. Cancer Res; 73(1); 160–71. ©2012 AACR.