Wolfram, Joy

ABSTRACT Cellular senescence in the kidney plays a crucial role in the progression of acute kidney injury and chronic kidney disease. Therapeutic approaches targeting senescent cells, such as small molecule senolytic and senomorphic drugs, display efficacy in preclinical models. However, such drugs pose a risk of adverse effects and only partially mitigate disease progression, highlighting the need for new therapeutic approaches that more comprehensively and safely address disease pathways in aging kidney disease. This review discusses the potential of extracellular vesicles and longevity proteins, such as α‐klotho and silencing information regulator 2‐related enzyme 1 (SIRT1), in regulating cellular senescence and alleviating kidney fibrosis. Particularly, combination therapy that simultaneously targets inflammation, tissue damage, and senescence is promising for kidney disease, given the potential to synergistically overcome the limitations of current unimodular treatment modalities and pave the way for more effective management of kidney disease. This review highlights the mechanisms of cellular senescence in kidney disease, particularly in diabetic kidney disease, the latest knowledge on senotherapy, and the potential and challenges of new therapeutic strategies, including combining extracellular vesicles and longevity proteins.

Viral myocarditis is an inflammatory cardiomyopathy with no pharmacologic agent currently able to quell the immune mechanisms that damage the heart in their inflammatory crossfire to quell infections, without risk of immunocompromise. Sex differences are well known in inflammatory and/or infectious diseases. Females display more efficient, anti-inflammatory responses to external threats like viruses, while males launch a pro-inflammatory defensive response. Myocarditis is a classic case of an infectious, inflammatory disease being more frequent and more severe in men, while pre-menopausal women are most protected. Extracellular vesicles are biocompatible, highly abundant, stable messaging carriers with no transformative risk, and hold great promise as therapeutics due to their functional cargo. As EVs are soluble mediators of the conversations between cells, we seek to redirect protective anti-inflammatory messaging from pre-menopausal women to male mice, using a highly translational model of murine myocarditis. We derive EVs from omental fat, repurposing this source to derive a purified, therapeutic component. Our results show robust immune system control, with EV treatments effectively attenuating pro-inflammatory inflammasome and complement markers, while leaving regulatory components like M2b macrophages and complement component CR1 unchanged. Treated mice are healthier during acute disease and demonstrate better heart function than control mice, despite being a strain susceptible to myocarditis and subsequent dilated cardiomyopathy. Tests using EVs from similarly aged men do not demonstrate this anti-inflammatory capacity. Proteomic and RNA-sequencing content comparisons of these EVs from these two donor groups reveal important proteins and regulatory micro RNAs. Our data suggest that EVs from fat may be a novel anti-inflammatory treatment for viral myocarditis, however individual donors show varying therapeutic capacity, with EV content heavily influenced by sex and age. This study not only addresses a major therapeutic gap in the myocarditis field, but also provides functional and -omic based comparisons of EVs from donors differing only by sex, to test if female cardioprotection from myocarditis is transferrable.

AbstractPatients with viral myocarditis are at risk of sudden death and may progress to dilated cardiomyopathy (DCM). Currently, no disease‐specific therapies exist to treat viral myocarditis. Here it is examined whether reconstituted, lyophilized extracellular vesicles (EVs) from platelets from healthy men and women reduce acute or chronic myocarditis in male mice. Human‐platelet‐derived EVs (PEV) do not cause toxicity, damage, or inflammation in naïve mice. PEV administered during the innate immune response significantly reduces myocarditis with fewer epidermal growth factor (EGF)‐like module‐containing mucin‐like hormone receptor‐like 1 (F4/80) macrophages, T cells (cluster of differentiation molecules 4 and 8, CD4 and CD8), and mast cells, and improved cardiac function. Innate immune mediators known to increase myocarditis are decreased by innate PEV treatment including Toll‐like receptor (TLR)4 and complement. PEV also significantly reduces perivascular fibrosis and remodeling including interleukin 1 beta (IL‐1β), transforming growth factor‐beta 1, matrix metalloproteinase, collagen genes, and mast cell degranulation. PEV given at days 7–9 after infection reduces myocarditis and improves cardiac function. MicroRNA (miR) sequencing reveals that PEV contains miRs that decrease viral replication, TLR4 signaling, and T‐cell activation. These data show that EVs from the platelets of healthy individuals can significantly reduce myocarditis and improve cardiac function.

Extracellular vesicles (EVs) play important roles in (patho)physiological processes by mediating cell communication. Although EVs contain glycans and glycosaminoglycans (GAGs), these biomolecules have been overlooked due to technical challenges in comprehensive glycome analysis coupled with EV isolation. Conventional mass spectrometry (MS)-based methods are restricted to the assessment of N-linked glycans. Therefore, methods to comprehensively analyze all glyco-polymer classes on EVs are urgently needed. In this study, tangential flow filtration-based EV isolation was coupled with glycan node analysis (GNA) as an innovative and robust approach to characterize most major glyco-polymer features of EVs. GNA is a molecularly bottom-up gas chromatography-MS technique that provides unique information that is unobtainable with conventional methods. The results indicate that GNA can identify EV-associated glyco-polymers that would remain undetected with conventional MS methods. Specifically, predictions based on GNA identified a GAG (hyaluronan) with varying abundance on EVs from two different melanoma cell lines. Enzyme-linked immunosorbent assays and enzymatic stripping protocols confirmed the differential abundance of EV-associated hyaluronan. These results lay the framework to explore GNA as a tool to assess major glycan classes on EVs, unveiling the EV glycocode and its biological functions.

Cancer stem cells (CSC) within non–small cell lung carcinoma (NSCLC) tumors drive NSCLC progression, metastasis, relapse, and intrinsic chemoresistance. Understanding the mechanisms that support the malignant phenotypes of NSCLC CSCs may provide insights for improved NSCLC therapeutic interventions. Here, we report that expression of RAB27B, a small GTPase, is significantly upregulated in NSCLC CSCs when compared with bulk cancer cells (BCC). Short hairpin RNA–mediated knockdown of RAB27B leads to a loss of stem cell marker gene expression and reduced NSCLC spheroid growth, clonal expansion, transformed growth, invasion, and tumorigenicity. We find that NSCLC CSCs secrete significantly more extracellular vesicles (EV) than BCCs, and that this is RAB27B-dependent. Furthermore, CSC-derived EVs, but not BCC-derived EVs, induce spheroid growth, clonal expansion, and invasion in BCCs. Finally, RAB27B is required for CSC-derived EV-induced stemness in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating, invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Our findings further suggest that inhibition of RAB27B-dependent EV secretion may be a potential therapeutic strategy for NSCLC. Significance: Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.

Abstract Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for ~85% of all lung cancer diagnoses. A sub-population of cells within human NSCLC tumors called cancer stem cells (CSCs) have been found to exhibit distinct properties that contribute to factors associated with poor outcomes in NSCLC patients including metastasis, relapse, and therapeutic resistance. We previously performed RNAseq analysis on bulk cancer cells (BCCs) and CSCs from a panel of primary NSCLC tumors and cell lines to identify signaling mechanisms involved in the tumorigenic behavior of NSCLC CSCs. Our analysis revealed that expression of RAB27B, a small GTPase that plays a critical role in extracellular vesicle (EV) formation and secretion, was significantly upregulated in CSCs when compared to BCCs. Interestingly, RAB27B is overexpressed and predicts patient survival in NSCLC. The goal of this study was to assess the role of RAB27B in the tumorigenic behavior of NSCLC CSCs and determine the mechanism by which RAB27B promotes an aggressive stem-cell like phenotype in NSCLC cells an aspect of RAB27B function that has not been investigated in NSCLC. QPCR and immunoblot analysis was used to compare RAB27B mRNA and protein expression in a panel of NSCLC BCCs and CSCs. Two independent shRNA constructs were used to knockdown (KD) RAB27B in NSCLC CSCs. The effects of RAB27B KD on CSC expansion, transformed growth and invasion were analyzed by clonal expansion, soft agar colony formation and Boyden chamber assays, respectively. Xenograft and tail vein mouse models were used to assess the effects of RAB27B KD on CSC tumor growth and lung colonization. EVs were isolated from the conditioned media of BCCs, CSCs and RAB27B KD CSCs using differential ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting according to MISEV guidelines. Our analysis revealed that RAB27B is significantly upregulated in NSCLC CSCs compared to BCCs. RAB27B KD cells exhibited a significant decrease in clonal expansion, transformed growth and invasion when compared to non-target shRNA control CSCs. In vivo studies demonstrated that RAB27B is important for the tumorigenicity of CSCs and promotes tumor cell proliferation, and angiogenesis. EVs were positive for EV-specific markers and negative for an intracellular vesicle marker. CSCs secrete significantly more EVs when compared to BCCs and KD of RAB27B in CSCs led to a decrease in the levels of EVs secreted. Furthermore, delivery of CSC-derived, but not BCC-derived EVs to BCCs induced spheroid growth, clonal expansion, and invasion in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating and invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Citation Format: Kayleah M. Meneses, Jennifer A. Lindemann, Prita Pandya, Dania S. Al-Qasrawi, Ryan Argo, Celeste Weems, Danielle J. Beetler, Irene K. Yan, Joy Wolfram, Tushar C. Patel, Verline Justilien. RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2444.

AbstractExtracellular vesicles (EVs) are cell‐released, heterogenous nanoparticles that play important roles in (patho)physiological processes through intercellular communication. EVs are often depicted as having a single lipid bilayer, but many studies have demonstrated the existence of multilayered EVs. There has been minimal inquiry into differences between unilamellar and multilamellar EVs in terms of biogenesis mechanisms and functional effects. This commentary speculates on potential causes and roles of multilamellar EVs and serves as a call to action for the research community to unravel the complex layers of EVs.

AbstractWith an exponential increase in extracellular vesicle (EV) studies in the past decade, focus has been placed on standardization of experimental design to ensure inter‐study comparisons and validity of conclusions. In the case of in vitro assays, the composition of cell culture media is important to consider for EV studies. In particular, levels of lipoproteins, which are critical components of the interstitial fluid, should be taken into consideration. Results from this study reveal that lipoprotein levels in cell culture medium impact the effects that EVs have on recipient cells. Additionally, evidence of EV binding and fusion to lipoprotein‐like structures in plasma is provided. However, it is unclear whether the impact of lipoproteins in cell culture is due to direct interactions with EVs, indirect effects, or a combination of both mechanisms. Taken together, cell culture studies performed in the absence of physiological levels of lipoproteins are unlikely to reflect interactions that occur between EVs and recipient cells in an in vivo environment.