Angom, Ramcharan Singh

Recent advancements in disease modeling for myocardial infarction (MI) and coronary artery disease (CAD) have significantly enhanced our understanding of cardiovascular pathology and therapeutic development. This review comprehensively reviews the integration of in vivo and in vitro approaches to better model and study these conditions. We specifically focus on cutting-edge in vitro techniques, such as cardiac organoids, engineered heart tissues, and patient-derived induced pluripotent stem cells (iPSCs), which allow for detailed exploration of cellular and molecular mechanisms involved in MI and CAD. These models provide insights into ischemic injury, myocardial remodeling, and the effects of potential therapeutic interventions at a cellular level. In parallel, we discuss advances in the in vivo models, including genetically modified mice and large animal models, which offer valuable information on disease progression, cardiac function, and response to treatments within a more complex physiological context. By bridging these in vivo and in vitro approaches, researchers can gain a more comprehensive understanding of disease mechanisms, validate experimental findings, and accelerate the development of effective therapies. This review highlights recent progress, identifies current limitations, and proposes strategies for future research to enhance the translation of model-based discoveries into clinical practice for MI and CAD.

Melanoma is the most aggressive subtype of cancer, with a higher propensity to spread compared to most solid tumors. The application of OMICS approaches has revolutionized the field of melanoma research by providing comprehensive insights into the molecular alterations and biological processes underlying melanoma development and progression. This review aims to offer an overview of melanoma biology, covering its transition from primary to malignant melanoma, as well as the key genes and pathways involved in the initiation and progression of this disease. Utilizing online databases, we extensively explored the general expression profile of genes, identified the most frequently altered genes and gene mutations, and examined genetic alterations responsible for drug resistance. Additionally, we studied the mechanisms responsible for immune checkpoint inhibitor resistance in melanoma.

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.

The zebrafish (Danio rerio) has emerged as an appreciated and versatile model organism for studying cardiovascular and metabolic diseases, offering unique advantages for both basic research and drug discovery. The genetic conservation between zebrafish and humans and their high fecundity and transparent embryos allow for efficient large-scale genetic and drug-oriented screening studies. Zebrafish possess a simplified cardiovascular system that shares similarities with mammals, making them particularly suitable for modeling various aspects of heart development, function, and disease. The transparency of zebrafish embryos enables the real-time visualization of cardiovascular dynamics, offering insights into early embryonic events and facilitating the study of heart-related anomalies. In metabolic research, zebrafish provide a cost-effective platform for modeling obesity, type 2 diabetes, hyperlipidemia, and other metabolic disorders. Their high reproductive rate allows for the generation of large cohorts for robust statistical analyses, while advanced genetic tools, such as CRISPR/Cas9, enable precise gene editing with which to model specific genetic mutations associated with human diseases. Zebrafish metabolic models have been instrumental in elucidating the molecular mechanisms underlying metabolic diseases, studying the effects of environmental factors, and identifying potential therapeutic targets. Additionally, the permeability of zebrafish embryos to small molecules facilitates drug discovery and screening, offering a rapid and economical approach to identifying compounds with therapeutic potential. In conclusion, zebrafish cardiovascular and metabolic disease models continue to contribute significantly to our perception of disease pathogenesis, providing a platform for translational research and developing novel therapeutic interventions. The versatility, scalability, and genetic manipulability of zebrafish position them as an invaluable asset in unraveling the complexities of cardiovascular and metabolic diseases. This review presents an overview of the zebrafish model’s key features and contributions to investigating cardiovascular and metabolic disorders. We discuss the benefits and drawbacks of using zebrafish models to study human disease and the critical findings revealed by the progress in this endeavor to date.

Abstract Background Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. Experimental Design: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. Results EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. Conclusion Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Glioblastoma multiforme (GBM) is a primary malignant brain tumor characterized by a high grade of malignancy and an extremely unfavorable prognosis. The current efficacy of established treatments for GBM is insufficient, necessitating the prompt development of novel therapeutic approaches. The progress made in the fundamental scientific understanding of GBM is swiftly translated into more advanced stages of therapeutic studies. Despite extensive efforts to identify new therapeutic approaches, GBM exhibits a high mortality rate. The current efficacy of treatments for GBM patients is insufficient due to factors such as tumor heterogeneity, the blood–brain barrier, glioma stem cells, drug efflux pumps, and DNA damage repair mechanisms. Considering this, pharmacological cocktail therapy has demonstrated a growing efficacy in addressing these challenges. Towards this, various forms of immunotherapy, including the immune checkpoint blockade, chimeric antigen receptor T (CAR T) cell therapy, oncolytic virotherapy, and vaccine therapy have emerged as potential strategies for enhancing the prognosis of GBM. Current investigations are focused on exploring combination therapies to mitigate undesirable side effects and enhance immune responses against tumors. Furthermore, clinical trials are underway to evaluate the efficacy of several strategies to circumvent the blood–brain barrier (BBB) to achieve targeted delivery in patients suffering from recurrent GBM. In this review, we have described the biological and molecular targets for GBM therapy, pharmacologic therapy status, prominent resistance mechanisms, and new treatment approaches. We also discuss these promising therapeutic approaches to assess prospective innovative therapeutic agents and evaluated the present state of preclinical and clinical studies in GBM treatment. Overall, this review attempts to provide comprehensive information on the current status of GBM therapy.

Introduction: Genome-wide association studies (GWAS) have discovered >300 associations for CAD. Few loci are functionally characterized and may represent new mechanisms of disease. Hypothesis: Can an unbiased transcriptional screen for the >2000 candidate GWAS genes in endothelial cells (ECs) identify causal genes and disease-relevant pathways at CAD GWAS loci. Methods: We applied CRISPRi-Perturb-seq to knock down expression of genes within 500 Kb of coronary artery disease GWAS loci (2,300 genes in total) and measure their transcriptomic effects using single-cell RNA-seq. Validation of candidate causal genes was conducted in primary human ECs and zebrafish. Results: We identified 50 programs of co-expressed genes, which represent core cellular pathways (i.e. ribosome biogenesis) and 13 EC-specific pathways such as flow response and angiogenesis. The EC-specific programs show the greatest contribution to CAD heritability. 5 EC-specific programs have the greatest number of GWAS candidate genes. We find that 43 of 306 CAD GWAS signals converge onto these 5 gene programs, and all are regulated by genes in the cerebral cavernous malformations (CCM) pathway—which coordinates KLF2/4 transcriptional responses in endothelial cells, but has not been linked to CAD risk. The strongest regulator of these programs is TLNRD1 , which we show is a new CAD gene and novel regulator of the CCM pathway. TLNRD1 loss-of-function alters actin organization and barrier function in endothelial cells in vitro , and heart development in zebrafish in vivo . Conclusions: High-throughput functional analysis of 2,300 genes proximal to CAD GWAS loci prioritized pathways—such as angiogenesis and EC migration—that are regulated by multiple risk SNPs. Our study identifies new genes that likely influence risk for CAD, identifies convergence of CAD genes into certain pathways in endothelial cells, and prioritizes study of genes in the CCM-signaling pathway for contribution to CAD risk.

BACKGROUND: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) technology-mediated genome editing has significantly improved the targeted inactivation of genes in vitro and in vivo in many organisms. Neuropilins play crucial roles in zebrafish heart regeneration, heart failure in mice, and electrical remodeling after myocardial infarction in rats. But the cell-specific functions of nrp1 have not been described before. In this study, we have investigated the role of nrp1 isoforms, including nrp1a and nrp1b , in cardiomyocytes during cardiac injury and regeneration in adult zebrafish hearts. METHODS: In this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specific cmlc2 promoter drives Cas9 expression to silence the nrp1 gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes a unique tool to regulate the gene knockout in both the developmental and adult stages and hence widens the possibility of loss-of-function studies in zebrafish at different stages of development and adulthood. Using this approach, we investigated the role of neuropilin isoforms nrp1a and nrp1b in response to cardiac injury and regeneration in adult zebrafish hearts. RESULTS: We observed that both the isoforms ( nrp1a and nrp1b ) are upregulated after the cryoinjury. Interestingly, the nrp1b knockout significantly delayed heart regeneration and impaired cardiac function in the adult zebrafish after cryoinjury, demonstrated by reduced heart rate, ejection fractions, and fractional shortening. In addition, we show that the knockdown of nrp1b but not nrp1a induces activation of the cardiac remodeling genes in response to cryoinjury. CONCLUSIONS: To our knowledge, this study is novel where we have reported a heat-shock–mediated conditional knockdown of nrp1a and nrp1b isoforms using CRISPR/Cas9 technology in the cardiomyocyte in zebrafish and furthermore have identified a crucial role for the nrp1b isoform in zebrafish cardiac remodeling and eventually heart function in response to injury.

CRISPR/Cas9 technology-mediated genome editing has significantly improved inactivation of genes both in vitro and in vivo in many organisms in a targeted manner. This study reports a novel CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. In this vector, the cardiac-specific, cardiac myosin light chain 2 ( cmlc2) promoter drives the Cas9 expression to silence the neuropilin1(nrp1) gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes an inimitable tool to regulate the gene knockout in both the developmental and adult stages. Hence, it widens the possibility of loss-of-function studies in zebrafish at different stages of development. In contrast to mammals, adult zebrafish hearts can regenerate after an injury. Neuropilins (nrp) are co-receptors that have been reported earlier to play crucial roles in zebrafish heart regeneration. Using this novel tool, we investigated the role of neuropilin isoforms, nrp1a and nrp1b, in response to cardiac injury and regeneration in adult zebrafish hearts. We observed that both the isoforms ( nrp1a and nrp1b ) are upregulated in the injured heart. Interestingly, the nrp1b -knockout significantly altered heart regeneration and reduced heart function, including heart rate, ejection fraction, and fractional shortening in the adult zebrafish. Moreover, the knockdown of nrp1b , but not nrp1a, further induces the cardiac remodeling genes activation in response to cryoinjury. These results identify a crucial role for nrp1b isoform in zebrafish heart function. To our knowledge, this is the first study where we have reported a heat shock-mediated conditional knockdown of nrp1a and nrp1b isoforms in the heart by using CRISPR/Cas9 technology and reveal a new molecular role of neuropilins in cardiac regeneration.

AbstractBiologically derived nanoparticles such as extracellular vesicles are promising candidates for therapeutic applications. In vivo toxicity of biological nanoparticles can result in tissue or organ damage, immunological perturbations, or developmental effects but cannot be readily predicted from in vitro studies. Therefore, an essential component of the preclinical assessment of these particles for their use as therapeutics requires screening for adverse effects and detailed characterization of their toxicity in vivo. However, there are no standardized, comprehensive methods to evaluate the toxicity profile of nanoparticle treatment in a preclinical model. Here, we first describe a method to prepare bovine milk‐derived nanovesicles (MNVs). These MNVs are inexpensive to isolate, have a scalable production platform, and can be modified to achieve a desired biological effect. We also describe two vertebrate animal models, mice and zebrafish, that can be employed to evaluate the toxicity profile of biologically derived nanoparticles, using MNVs as an example. Treatment‐induced organ toxicity and immunological effects can be assessed in mice receiving systemic injections of MNVs, and developmental toxicity can be assessed in zebrafish embryos exposed to MNVs in embryo water. Utilizing these animal models provides opportunities to analyze the toxicity profiles of therapeutic extracellular vesicles in vivo. © 2021 Wiley Periodicals LLC.Basic Protocol 1: Preparation of milk‐derived nanovesiclesBasic Protocol 2: In vivo screening for organ toxicity and immune cell profiling using miceBasic Protocol 3: In vivo developmental toxicity screening using zebrafish