Wang, Enfeng

Introduction Recent transcriptome analysis has demonstrated increased expression of Vascular Endothelial Growth Factor receptor-1 (VEGFR-1/FLT1) and in AD brain. Increased expression of VEGFR1 and its ligand VEGFB were associated with a more rapid rate of cognitive decline, providing evidence of a potential link between increased VEGFR-1 expression in AD pathogenesis. In this study, we explored the potential role of VEGFR-1 expression in neurons on AD pathology. Methods To confirm VEGFR1 expression in AD brains, we first performed immunostaining in AD brain sections (AD - Braak stage V-VI, and normal controls - Braak 0-II). And to determine a potential detrimental role of neuronal VEGFR1 expression on AD associated pathologies, we exposed SH-SY5Y human neuroblastoma cells and mouse primary neurons to either hypoxia conditions (1%O 2 ) or 5 μ Aβ1-42 oligomers or fibrils for 24, 28 and 72hrs. Results In this study, we found preferential staining of VEGFR-1 in the neuropil and neuronal cell bodies both in AD and Control hippocampus and increased VEGFR-1 immunoreactivity in dystrophic neuritic processes in the vicinity of Thio-S positive amyloid plaques in AD brains. And treatment of SH-SY5Y human neuroblastoma cell line and mouse primary neurons, with either hypoxia conditions or Aβ1-42 oligomers, resulted in increased VEGFR-1 expression and cleaved caspase 3 activation, leading to neuronal toxicities/cell death. Similarly, treatment with Aβ1-42 fibrils also increased VEGFR-1 and cleaved caspase 3 protein levels in the SH-SY5Y cells whereas treatment with Aβ1-42 monomers had no effect on VEGFR-1 expression. In addition, we show that over-expression of VEGFR-1 intracellular domains in SH-SY5Y cells directly induced neuronal toxicities and importantly, siRNA-mediated knockdown of VEGFR-1 in neurons prevented the hypoxia, Aβ1-42 oligomer and Aβ1-42 fibril-induced toxicities and cell death phenotypes. Treatment with either hypoxia or Aβ1-42 oligomers also reduced expression of cell survival genes including VEGFR-2 and Hippo pathway YAP1 and siRNA-mediated VEGFR-1 knockdown in the neurons normalized expression of both VEGFR-2 and YAP1. Using differential gene expression analysis, we demonstrated upregulation of several inflammatory/interferon-stimulated genes (ISGs) as well as increased expression of genes involved in activation of oxidative stress and cell death pathways in response to Aβ1-42 oligomers treatment in mouse primary neurons. And siRNA-mediated VEGFR-1 knockdown in the mouse primary neurons, reduced gene expression of both the ISGs and oxidative stress/cell death pathways in response to Aβ1-42 oligomer treatment. Discussion In summary, these results show that siRNA-mediated knockdown of VEGFR-1 in neurons significantly prevented hypoxia, Aβ1-42 oligomer and Aβ1-42 fibril-induced cellular toxicities and cell death phenotypes, indicating a potential detrimental role of aberrant VEGFR-1 expression and signaling in response to AD associated pathologies.

Abstract Background Glioblastoma (GBM) is the most common high-grade primary malignant brain tumor, characterized by a notably poor prognosis. Current treatments for GBM have shown limited effectiveness in improving patient survival, highlighting the urgent need for novel therapeutic strategies. Combination therapy offers significant potential in overcoming resistance by targeting multiple signaling pathways; however, it often comes with increased toxicity compared to monotherapy. Co-encapsulating multiple therapeutic agents into a tumor-targeted drug delivery platform holds promise for overcoming these limitations and improving treatment outcomes. Methods We developed a tumor-targeted liposomal nanoformulation (TTL) using phospholipids, cholesterol, DSPE-(PEG)2000-OMe, and a proprietary tumor-targeting peptide (TTP). The TTL was loaded with everolimus (TTL-E), vinorelbine (TTL-V), rapamycin (TTL-R), a combination (TTL-EV), or (TTL-RV). These formulations were tested in vivo on orthotopic GBM mice, combined with temozolomide and radiation. RNA sequencing was performed to identify molecular and transcriptome changes post-treatment. Results TTL demonstrated tumor-specific uptake, effectively delivering drugs to GBM tumors. TTL-EV and TTL-RV outperformed single-drug formulations. Radiation combined with TTL-EV/RV improved tumor growth inhibition and survival, while temozolomide provided minimal benefit. Transcriptome analysis revealed differentially expressed genes (DEGs) linked to DNA damage repair, cell cycle, metabolism, and extracellular matrix pathways. Conclusion TTL crossed the blood-brain barrier, targeting tumors effectively. Radiation plus TTL-EV/RV enhanced tumor suppression and survival in GBM models. Gene expression analysis identified DEGs related to DNA damage and cell death. Mechanistic studies suggest TTL-EV plus radiation inhibits mTOR/MAPK pathways and sensitizes tumors to radiation. These findings offer a potential approach for improving GBM treatment.

Myocardial infarction (MI) remains a leading cause of mortality worldwide, yet effective cardioprotective strategies remain limited in clinical settings. Vascular endothelial growth factor B (VEGFB) has emerged as a promising therapeutic candidate in MI, but the role of its co-receptor, Neuropilin-1 (NRP1), in cardiomyocyte (CM) survival under ischemic stress remains poorly understood. Here, we investigated VEGFB-NRP1 signaling using an in vivo zebrafish model of cardiac injury as well as in vitro hypoxia models in CMs. We demonstrated that VEGFB overexpression conferred protection against ischemic injury and enhanced cardiac regeneration in the zebrafish heart. Mechanistically, we showed that VEGFB treatment enhances CM viability through reducing reactive oxygen species (ROS), ferroptosis activation, and preserving mitochondrial integrity. We also demonstrated that NRP1 knockdown in the CMs abolished the VEGFB-mediated protective effects, indicating the significant role of NRP1 signaling in VEGFB-induced cardioprotective effects in MI. Lastly, using transcriptome analysis, we confirmed that VEGFB induces anti-apoptotic and anti-ferroptosis gene programs in CMs in response to hypoxic stress. Collectively, our findings provide mechanistic insight into cell death activation pathways, including ferroptosis, in response to ischemic stress and further validate the therapeutic potential of VEGFB in promoting CM survival in ischemic heart disease.

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, characterized by aggressive tumor biology, poor vascularization, dense stromal barriers, and profound resistance to chemotherapy. GAIP-interacting protein C-terminus 1 (GIPC1), a PDZ-domain-containing adaptor protein, is highly overexpressed in PDAC and plays a critical role in tumor progression and chemoresistance. This study aimed to develop and evaluate a novel tumor-targeted liposomal siRNA delivery system (LGIPCsi) to silence GIPC1 and enhance the therapeutic efficacy of gemcitabine (GEM) in PDAC; Methods: LGIPCsi nanoparticles were synthesized and optimized for physicochemical stability, siRNA complexation efficiency, and tumor-targeting capability. Their therapeutic efficacy was assessed using in vitro pancreatic cancer cell models and in vivo orthotopic and patient-derived xenograft (PDX) models of PDAC. Biodistribution, tumor uptake, and antitumor efficacy were evaluated following systemic administration. Combination studies were performed to assess the synergistic effects of LGIPCsi and GEM; Results: GIPC1 silencing significantly sensitized pancreatic cancer cells to GEM, resulting in enhanced inhibition of tumor cell proliferation in vitro. In vivo, systemic administration of LGIPCsi achieved efficient intratumoral delivery of siGIPC1, leading to marked tumor growth suppression. Combination therapy with GEM and LGIPCsi produced synergistic antitumor effects, with substantial tumor regression compared to monotherapy groups. Importantly, no significant systemic toxicity was observed in treated animals; Conclusions: This study identifies GIPC1 as a promising therapeutic target in PDAC and demonstrates that tumor-targeted siRNA nanomedicine can effectively overcome chemoresistance when combined with standard chemotherapy. The LGIPCsi platform offers a rational and translational strategy to enhance treatment efficacy in PDAC through targeted RNAi-based combination therapy.

Abstract Background Myocardial infarction (MI) remains a leading cause of mortality worldwide. Recent studies suggest a cardioprotective role for vascular endothelial growth factor-B (VEGF-B) in MI. However, the molecular mechanisms of VEGF-B-mediated signaling via its co-receptor Neuropilin-1 (NRP1) in MI are poorly understood. In this study, we investigated the intricate signaling mechanisms involving VEGF-B and NRP1 in cardiomyocytes (CMs) using ischemic injury as a model of MI. And secondly, we further validated the protective role of VEFG-B in ischemic heart disease, shedding light on their roles not only in cardiac function but also in its therapeutic potential. Methods In this study, we utilized both in vitro and in vivo approaches to elucidate the role of VEGF-B and NRP1 signaling in MI and how it manifests a protective role in mitochondrial functions and cardiac regeneration following ischemic injury. We used two different cardiomyocyte cell lines, H9c2 (rat ventricular cardiomyocytes) and HL-1 (mouse ventricular cardiomyocytes), and induced hypoxia conditions, using either 1% oxygen or 200µM cobalt chloride (CoCl 2 ) to mimic the myocardial infarction-induced ischemic injury in the heart. In addition, we developed a novel heat shock inducible zebrafish model of a cardiomyocyte-specific VEGF-B overexpression system to further examine the protective role of VEGF-B in vivo . Results Our findings indicate that both VEGF-B and NRP1 are predominantly expressed in heart tissue compared to other tissues, and their expression is altered in response to hypoxia/ischemic injury. Our results demonstrate that VEGF-B treatment prior to hypoxia enhances cardiomyocyte survival, while NRP1 knockdown abolishes this protective effect, highlighting a prominent role of NRP1 signaling in VEGF-B-mediated cardio protection. Furthermore, we found that VEGF-B promotes cardiomyocyte survival by improving mitochondrial function, as evidenced by reduced oxidative stress and ROS accumulation, decreased oxidative stress, preserved mitochondrial membrane potential, and increased ATP levels. Lastly, using our VEGF-B transgenic zebrafish model, we demonstrated that VEGF-B overexpression in zebrafish cardiomyocytes protects the heart from ischemic injury and enhances cardiac regeneration in an NRP1-dependent manner. Conclusion Our study has uncovered an important role of VEGF-B-NRP1 signaling axis in VEGF-B mediated cell survival and in beneficial mitochondrial functions in the CMs. Importantly, we also demonstrated that VEGF-B is protective against ischemic injury in vivo using a novel zebrafish model Novelty and Significance What Is Known? Myocardial infarction (MI) results in mitochondrial dysfunction, cardiomyocyte death, and adverse ventricular remodeling. Vascular Endothelial Growth Factor B (VEGF-B) is traditionally associated with cardiac metabolism, survival, vascular biology, particularly in angiogenesis and lipid metabolism. The neurophilin-1 (NRP1) receptor, a co-receptor for VEGF-B, is expressed in cardiomyocytes and implicated in mitochondrial signaling pathways. Emerging evidence suggests that VEGF-B may influence mitochondrial integrity and function under stress conditions. What New Information Does This Article Contribute? VEGF-B exerts a protective effect on cardiomyocyte mitocondria following ischemic injury by preserving mitochondrial function and reducing oxidative stress. NRP1 receptor as a key mediator of VEGF-B modulates mitochondrial homeostasis in cardiomyocytes. VEGF-B signaling can attenuate apoptosis and enhance mitochondrial bioenergetics post-MI, suggesting a novel cardioprotective mechanism. Clinical Implication: These findings suggest that therapeutic strategies aimed at enhancing VEGF-B/NRP1 signaling may improve mitochondrial function and cardiac recovery after MI, offering a novel avenue for limiting heart failure progression in ischemic heart disease.

Abstract Background: TTFields are non-invasive and novel treatment modality that utilizes low intensity, alternating, and intermediate frequency electrical fields to inhibit cancer cell proliferation through a multitude of mechanisms. These include inhibition of mitosis, disruption of cancer cell membranes, metabolomic dysregulation of cancer cells, and immunogenic activation of the tumor microenvironment (TME). TTFields are approved in glioblastoma multiforme, non-small cell lung cancer and malignant mesothelioma. The PANOVA2 trial demonstrated promising efficacy for TTFields when applied together with chemotherapy in patients with PDAC. Mechanisms of TTFields in PDAC need further delineation. We sought to investigate the efficacy of TTFields in vitro and in vivo in PDAC cell lines and mouse models. Preclinical studies were conducted in concert with an going clinical trial (NCT04605913). Experimental Procedures: Utilizing the inovitro apparatus we studied the effect of TTFields (150kHz, intensity 2.9+0.2 V cm RMS, 37ċ, over 72 hrs) in an array of PDAC (Panc1, KPC, Panc2, and AsPC1) and human pancreatic stellate cells (hPSC) cell lines, alone or together with cisplatin (Cis, 0.1μM) or gemcitabine (Gem, 0.01μM), compared to control. Cell viability analysis was conducted in each of the groups. In addition, cell lysates from Panc1 cells from concomitant treatment of TTFields plus Gem were probed for STAT3 and α-SMA to analyze stromal proteins and RAS pathway components (KRAS, P38, P42, and others) were performed via Westen Blot. We also performed RNA sequencing for the different groups to analyze gene expression levels. We further assessed the effect of TTFields (150kHz, intensity 1.8-2 V/cm RMS, over 2 weeks) using the inovivo system with and without chemotherapy (Cis, 0.25 mg/Kg per dose (i.p.) 3 times a week) compared to control in C57BL6 orthotopic murine models (6-7 weeks old C57BL6 mice). Summary of Results: Our analysis revealed statistically significant abrogation of PDAC cancer cell lines for the different groups and hPSC cell viability when compared to control for TTFields monotherapy (p <0.001) and TTFields concomitantly with Gem or Cis (p <0.0001). Western blot analysis demonstrated that TTFields alone and when combined with chemotherapy alters both the stroma and RAS pathways with confirmed downregulation of pSTAT3 and α-SMA and KRAS, P-P38, and P-P42. In vivo analysis revealed significant decrease in tumor volume and weight for the concomitant effect of TTFields + Cis. Conclusion: Our preclinical studies demonstrated the efficacy of TTFields as monotherapy and concomitantly with chemotherapy in PDAC and hPSC cell lines and mouse models. Mechanistic insights from the pre-clinical studies will inform the design of future TTFields studies in PDAC. Citation Format: Hani M. Babiker, Santanu Bhattacharya, Douaa Albelal, Shamit Kumar Dutta, Enfeng wang, Debabrata Mukhopadhyay. umor treating fields (TTFields) plus chemotherapy abrogates pancreatic ductal adenocarcinoma (PDAC) proliferation and alters the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1834.

Heart failure with preserved ejection fraction (HFpEF), characterized by cardiac diastolic dysfunction, leads to premature mortality with limited effective treatments. A recent study in HFpEF patients suggested neuropilin 1 (NRP1), an endothelial cell (EC) originated prognostic marker, might contribute to the pathogenesis of HFpEF. However, the functional role of NRP1 in the pathogenesis of HFpEF remains unclear. Purpose: To determine the functional role of NRP1 in the pathogenesis of HFpEF. Hypothesis: NRP-1 contributes to the pathogenesis of HFpEF, potentially mediated through chronic inflammation. Methods: HFpEF was induced by hypertensive and metabolic stresses in control (n=14) and tamoxifen-induced, endothelial cell-specific NRP1 knockout (NRP1 iEC-KO ) mice (n=13). We assessed cardiac diastolic function via echocardiography and pressure-volume loop through invasive catheters. To determine the inflammation level, we tested leukocyte adhesion, inflammatory marker vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) mRNA level in control and NRP1 shRNA knockdown (NRP1 KD) human umbilical vein endothelial cells (HUVECs) with a stimulation of TNF-α. Student t-test (two tailed) was used and the criterion level of significance was set at p<0.05. Results: We found a significantly improved diastolic function in NRP1 iEC-KO mice, evidenced by a significantly reduced end diastolic pressure in NRP1 iEC-KO mice (8±2mmHg; n=7) compared to the controls (17±2mmHg; n=9; p=0.01) after HFpEF induction. E/A ratio was also significantly lower in NRP1 iEC-KO mice (2.0±0.2; n=10) compared to controls (2.7±0.3; n=12; p=0.04). Moreover, the isovolumic relaxation time was also significantly reduced in NRP1 iEC-KO mice (18±0.8ms; n=13) compared to controls (20±0.8ms; n=14; p=0.03). In addition, In HUVECs, NRP1 KD significantly reduced inflammatory marker VCAM-1 mRNA expression (95±16; n=5) compared to controls (293±36; n=5; p<0.01) upon TNF-α treatment. Similarly, NRP1 KD significantly reduced ICAM-1 mRNA expression (44±14; n=5) compared to controls (83±9; n=5; p=0.03) upon TNF-α treatment. Furthermore, leukocyte adhesion was significantly lower in the NRP1 KD group (57.0 ±8.6 cells/field) compared to the control group (90.8 ±9.1 cells/field; p=0.04). Conclusion: Endothelial NRP1 contributes to cardiac diastolic dysfunction and the pathogenesis of HFpEF, potentially mediated through the activation of chronic low-grade inflammation.

Abstract Gold nanoparticles for therapeutic interventions have become a widely popular source due to the ease in surface chemistry available for modification. Several studies undertaken focusses on the size, shape of nanoparticles as well as the signaling mechanisms and yet, knowledge regarding the alteration in membrane dynamics such as nanomechanical properties and cytokines involved during their uptake has not been studies before. To overcome the knowledge deficit, we employed atomic force microscopy and qRT-PCR techniques to acquire nanomechanical attributes and cytokines expressions respectively, in Panc-1 and AsPC-1 cell lines treated with surface modified gold nanoparticles for 1- and 24-hours’ time point. In nanomechanics paradigm, impetus was given on both linear attributes such as membrane stiffness, deformation, and adhesion as well as non-linear attributes such as drained Poisson’s ratio, diffusion coefficient and pore size. On the other hand, qRT-PCR technique yielded alterations in relative mRNA expressions of pro-tumorigenic cytokines such as CCL2, CXCL1, CXCL2, CXCL3, IL8, IL11, IL18 and TGFβ-1. Further, using Pearson’s correlation, we deduced the dependency between nanomechanical properties and cytokines expressions. We evaluated several criteria such as receptor dependent vs independent, PEGylated vs non-PEGylated and 1 hr vs 24hrs, to deduce correlations between cytokines and nanomechanical attributes. In Panc-1 cell line, we observed that PEGylation vs non-PEGylation of gold nanoparticles demonstrated most significant correlations, whereas in AsPC-1 cell line, receptor independent mechanism played a pivotal role in establishing significant correlations. For instance, D_C and P_D were highly correlated with CXCL1, CXCL2 and TGFβ-1 when non-PEGylated gold nanoparticles were up taken by Panc-1 cells. Similarly, D_C and P_D were highly correlated consistently with our cytokine panel except for TGFβ-1. In AsPC-1 cell line, deformation was significantly correlated with CCL2, CXCL2, CXCL3 and IL-8 whereas, D_C and P_D were significantly correlated with CCL2, CXCL1, CXCL3, IL-18 and TGFβ-1 in receptor independent scenario. This study demonstrated that during the uptake of gold nanoparticles, non-linear nanomechanical attributes are detrimental over linear attributes. Moreover, Surface modified gold nanoparticles induce varying levels of alteration in pro-tumorigenic cytokines. Taken together, this study boosts our insights into correlations describing cytokines regulating nanomechanical attributes in endocytosis paradigm. Citation Format: Tanmay Kulkarni, Enfeng Wang, Ramcharan Singh Angom, Debabrata Mukhopadhyay, Santanu Bhattacharya. Insights into correlation between nanomechanical properties and cytokines expression during gold nanoparticle endocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 496.