Bone marrow-fibroblast progenitor cell-derived small extracellular vesicles promote cardiac fibrosis via miR-21-5p and integrin subunit αV signalling.

Cardiac fibrosis is the hallmark of cardiovascular disease (CVD), which is leading cause of death worldwide. Previously, we have shown that interleukin-10 (IL10) reduces pressure overload (PO)-induced cardiac fibrosis by inhibiting the recruitment of bone marrow fibroblast progenitor cells (FPCs) to the heart. However, the precise mechanism of FPC involvement in cardiac fibrosis remains unclear. Recently, exosomes and small extracellular vesicles (sEVs) have been linked to CVD progression. Thus, we hypothesized that pro-fibrotic miRNAs enriched in sEV-derived from IL10 KO FPCs promote cardiac fibrosis in pressure-overloaded myocardium. Small EVs were isolated from FPCs cultured media and characterized as per MISEV-2018 guidelines. Small EV's miRNA profiling was performed using Qiagen fibrosis-associated miRNA profiler kit. For functional analysis, sEVs were injected in the heart following TAC surgery. Interestingly, TGFß-treated IL10-KO-FPCs sEV increased profibrotic genes expression in cardiac fibroblasts. The exosomal miRNA profiling identified miR-21a-5p as the key player, and its inhibition with antagomir prevented profibrotic signalling and fibrosis. At mechanistic level, miR-21a-5p binds and stabilizes ITGAV (integrin av) mRNA. Finally, miR-21a-5p-silenced in sEV reduced PO-induced cardiac fibrosis and improved cardiac function. Our study elucidates the mechanism by which inflammatory FPC-derived sEV exacerbate cardiac fibrosis through the miR-21a-5p/ITGAV/Col1α signalling pathway, suggesting miR-21a-5p as a potential therapeutic target for treating hypertrophic cardiac remodelling and heart failure.

Challenges and future scope of exosomes in the treatment of cardiovascular diseases.

Exosomes are nanosized vesicles that carry biologically diverse molecules for intercellular communication. Researchers have been trying to engineer exosomes for therapeutic purposes by using different approaches to deliver biologically active molecules to the various target cells efficiently. Recent technological advances may allow the biodistribution and pharmacokinetics of exosomes to be modified to meet scientific needs with respect to specific diseases. However, it is essential to determine an exosome's optimal dosage and potential side effects before its clinical use. Significant breakthroughs have been made in recent decades concerning exosome labelling and imaging techniques. These tools provide in situ monitoring of exosome biodistribution and pharmacokinetics and pinpoint targetability. However, because exosomes are nanometres in size and vary significantly in contents, a deeper understanding is required to ensure accurate monitoring before they can be applied in clinical settings. Different research groups have established different approaches to elucidate the roles of exosomes and visualize their spatial properties. This review covers current and emerging strategies for in vivo and in vitro exosome imaging and tracking for potential studies.

UBE2T promotes breast cancer tumor growth by suppressing DNA replication stress.

Breast cancer is a leading cause of cancer-related deaths among women, and current therapies benefit only a subset of these patients. Here, we show that ubiquitin-conjugating enzyme E2T (UBE2T) is overexpressed in patient-derived breast cancer samples, and UBE2T overexpression predicts poor prognosis. We demonstrate that the transcription factor AP-2 alpha (TFAP2A) is necessary for the overexpression of UBE2T in breast cancer cells, and UBE2T inhibition suppresses breast cancer tumor growth in cell culture and in mice. RNA sequencing analysis identified interferon alpha-inducible protein 6 (IFI6) as a key downstream mediator of UBE2T function in breast cancer cells. Consistently, UBE2T inhibition downregulated IFI6 expression, promoting DNA replication stress, cell cycle arrest, and apoptosis and suppressing breast cancer cell growth. Breast cancer cells with IFI6 inhibition displayed similar phenotypes as those with UBE2T inhibition, and ectopic IFI6 expression in UBE2T-knockdown breast cancer cells prevented DNA replication stress and apoptosis and partly restored breast cancer cell growth. Furthermore, UBE2T inhibition enhanced the growth-suppressive effects of DNA replication stress inducers. Taken together, our study identifies UBE2T as a facilitator of breast cancer tumor growth and provide a rationale for targeting UBE2T for breast cancer therapies.

Management of inflammation in cardiovascular diseases.

Cardiovascular disease is the leading cause of morbidity and mortality world-wide. Recently, the role of inflammation in the progression of diseases has significantly attracted considerable attention. In addition, various comorbidities, including diabetes, obesity, etc. exacerbate inflammation in the cardiovascular system, which ultimately leads to heart failure. Furthermore, cytokines released from specialized immune cells are key mediators of cardiac inflammation. Here, in this review article, we focused on the role of selected immune cells and cytokines (both pro-inflammatory and anti-inflammatory) in the regulation of cardiac inflammation and ultimately in cardiovascular diseases. While IL-1ß, IL-6, TNFα, and IFNγ are associated with cardiac inflammation; IL-10, TGFß, etc. are associated with resolution of inflammation and cardiac repair. IL-10 reduces cardiovascular inflammation and protects the cardiovascular system via interaction with SMAD2, p53, HuR, miR-375 and miR-21 pathway. In addition, we also highlighted recent advancements in the management of cardiac inflammation, including clinical trials of anti-inflammatory molecules to alleviate cardiovascular diseases.

ALKBH5 Regulates SPHK1-Dependent Endothelial Cell Angiogenesis Following Ischemic Stress.

BACKGROUND: Endothelial cells dysfunction has been reported in many heart diseases including acute myocardial infarction, and atherosclerosis. The molecular mechanism for endothelial dysfunction in the heart is still not clearly understood. We aimed to study the role of m6A RNA demethylase alkB homolog 5 (ALKBH5) in ECs angiogenesis during ischemic injury. METHODS AND RESULTS: ECs were treated with ischemic insults (lipopolysaccharide and 1% hypoxia) to determine the role of ALKBH5 in ECs angiogenesis. siRNA mediated ALKBH5 gene silencing was used for examining the loss of function. In this study, we report that ALKBH5 levels are upregulated following ischemia and are associated with maintaining ischemia-induced ECs angiogenesis. To decipher the mechanism of action, we found that ALKBH5 is required to maintain eNOS phosphorylation and SPHK1 protein levels. ALKBH5 silencing alone or with ischemic stress significantly increased SPHK1 m6A mRNA methylation. In contrast, METTL3 (RNA methyltransferase) overexpression resulted in the reduced expression of SPHK1. CONCLUSION: We reported that ALKBH5 helps in the maintenance of angiogenesis in endothelial cells following acute ischemic stress via reduced SPHK1 m6A methylation and downstream eNOS-AKT signaling.

Decreased ascorbic acid biosynthesis in response to PMSG in the pre-pubertal female rat ovary.

Ascorbic acid (AA) is known to be an important antioxidant serving as a cofactor in collagen synthesis, and thus facilitates follicular growth in the ovary. Many studies have shown that AA is synthesized in the liver and transported to other organs including ovary, however, there is no direct evidence of ascorbic acid synthesis in the ovary. Hence, we examined the expression pattern of different proteins (SMP30/GNL and GULO) involved in the AA synthesis in pre-pubertal rat, which showed significant expression of these proteins, suggesting the synthesis of AA in the ovary. Accumulation of AA in the ovary during follicular growth has been well demonstrated. However, the effect of Pregnant Mare Serum Gonadotropin (PMSG) on the AA synthesis in the ovary has not been studied in detail. Hence to decipher the effect, different doses of PMSG were injected subcutaneously into the pre-pubertal female rats, and ovarian AA level was measured after 48 h. A significant increase in AA content was observed in PMSG treated animal groups. Further, to understand the mechanism underlying ovarian AA accumulation, the expression levels of SMP30/GNL and GULO genes were measured. Expression of both the genes was significantly suppressed, which suggested a lowered AA synthesis in the PMSG treated rat ovary. For further understanding, mRNA expression of AA transporters SVCT1 and SVCT2 encoded by SLC23A1 and SLC23A2 genes respectively were measured, which showed increased level of SVCT1 expression. These observations suggested that the increased AA content might not be due to increased synthesis of AA within the ovary but possibly due to increased uptake from blood during the stimulation of follicular growth.

Comparative analysis of the metal-dependent structural and functional properties of mouse and human SMP30.

Senescence Marker Protein (SMP30) is a metalloenzyme that shows lactonase activity in the ascorbic acid (AA) biosynthesis pathway in non-primate mammals such as a mouse. However, AA biosynthesis does not occur in the primates including humans. Several studies have shown the role of SMP30 in maintaining calcium homeostasis in mammals. In addition, it is also reported to have promiscuous enzyme activity with an organophosphate (OP) substrate. Hence, this study aims to recombinantly express and purify the SMP30 proteins from both mouse and human, and to study their structural alterations and functional deviations in the presence of different divalent metals. For this, mouse SMP30 (MoSMP30) as well as human SMP30 (HuSMP30) were cloned in the bacterial expression vector. Proteins were overexpressed and purified from soluble fractions as well as from inclusion bodies as these proteins were expressed largely in insoluble fractions. The purified proteins were used to study the folding conformations in the presence of different divalent cations (Ca2+, Co2+, Mg2+, and Zn2+) with the help of circular dichroism (CD) spectroscopy. It was observed that both MoSMP30 and HuSMP30 acquired native folding conformations. To study the metal-binding affinity, dissociation constant (Kd values) were calculated from UV-VIS titration curve, which showed the highest affinity of MoSMP30 with Zn2+. However, HuSMP30 showed the highest affinity with Ca2+, suggesting the importance of HuSMP30 in maintaining calcium homeostasis. Enzyme kinetics were performed with γ-Thiobutyrolactone and Demeton-S in the presence of different divalent cations. Interestingly, both the proteins showed lactonase activity in the presence of Ca2+. In addition, MoSMP30 and HuSMP30 also showed lactonase activity in the presence of Co2+ and Zn2+ respectively. Moreover, both the proteins showed OP hydrolase activities in the presence of Ca2+ as well as Zn2+, suggesting the metal-dependent promiscuous nature of SMP30.

Synthesis, biological evaluation and molecular docking study of 1-amino-2-aroylnaphthalenes against prostate cancer.

A series of functionalized naphthalene was synthesized and screened against human prostate cancer cell line (PC-3). The in vitro antiproliferative activity of the synthesized compounds was evaluated by monitoring their cytotoxic effects against PC-3 cells by using MTT assay. We observed that compound 5f resulted in more than 50% cell death at 14 µM. Treatment of PC-3 cells with 5f provides apoptosis by flow cytometry. Western blotting showed decreased expression of pro-caspase 8 and 9. Our study shows that cancer cell treated with 5f has higher concentration of reactive oxygen species as compare to untreated sample, which facilitate cancerous cell to enter apoptosis. Exact mechanism by which ROS is generated after 5f treatment is still under study. Molecular docking study further strengthens the results obtained from in vitro experiments. Compound 5f can be considered as a promising leads for anticancer agent against prostate cancer cells due to its potent cytotoxic activity and apoptotic effect.

In vitro synergistic effect of fluoroquinolone analogues in combination with artemisinin against Plasmodium falciparum; their antiplasmodial action in rodent malaria model.

BACKGROUND: Emergence of drug-resistant parasite strains has surfaced as a major obstacle in attempts to ameliorate malaria. Current treatment regimen of malaria relies on the concept of artemisinin-based combination therapy (ACT). METHODS: Fluoroquinolone analogues, compounds 10, 12 and 18 were investigated for their anti-malarial interaction in combination with artemisinin in vitro, against Plasmodium falciparum 3D7 strain, employing fixed-ratio combination isobologram method. In addition, the efficacy of these compounds was evaluated intraperitoneally in BALB/c mice infected with chloroquine-resistant Plasmodium berghei ANKA strain in the Peters' four-day suppressive test. RESULTS: Promising results were obtained in the form of synergistic or additive interactions. Compounds 10 and 12 were found to have highly synergistic interactions with artemisinin. Antiplasmodial effect was further verified by the convincing ED50 values of these compounds, which ranged between 2.31 and 3.09 (mg/kg BW). CONCLUSIONS: In vivo studies substantiated the potential of the fluoroquinolone derivatives to be developed as synergistic partners for anti-malarial drug combinations.