Lerman, Lilach O

Injection of A-MSC-derived EVs harvested from patients who are lean can resolve myocardial injury in mice with experimental renovascular hypertension more effectively than A-MSC-derived EVs from patients with obesity. These observations underscore and might have important ramifications for the self-healing capacity of patients with obesity and for the use of autologous EVs as a regenerative tool.

Autologous mesenchymal stem/stromal cells (MSCs) have demonstrated important therapeutic effects in several diseases. Cardiovascular risk factors may impair MSC mitochondrial structure and function, but the underlying mechanisms remain unknown. We hypothesized that metabolic syndrome (MetS) induces epigenetic alterations in mitochondria-related genes in swine MSCs. Pigs were fed a Lean or MetS diet (n = 6 each) for 16 weeks. MSCs were collected from subcutaneous abdominal fat, and DNA hydroxymethylation (5 hmC) profiles of mitochondria-related genes (MitoCarta-2.0) were analyzed by hydroxymethylated DNA immunoprecipitation and next-generation sequencing (hMeDIP-seq) in Lean- and MetS-MSCs untreated or treated with the epigenetic modulator vitamin (Vit)-C (n = 3 each). Functional analysis of genes with differential 5 hmC regions was performed using DAVID6.8. Mitochondrial structure (electron microscopy), oxidative stress, and membrane potential were assessed. hMeDIP-seq identified 172 peaks (associated with 103 mitochondrial genes) with higher and 416 peaks (associated with 165 mitochondrial genes) with lower 5 hmC levels in MetS-MSCs versus Lean-MSCs (≥2-fold, p < 0.05). Genes with higher 5 hmC levels in MetS + MSCs were primarily implicated in fatty acid metabolism, whereas those with lower 5 hmC levels were associated with electron transport chain activity. Vit-C increased 5 hmC levels in mitochondrial antioxidant genes, improved mitochondrial structure and membrane potential, and decreased oxidative stress. MetS alters 5 hmC levels of mitochondria-related genes in swine MSCs. Vit-C modulated 5 hmC levels in these genes and preserved mitochondrial structure and function in MetS-MSCs. These observations may contribute to development of strategies to overcome the deleterious effects of MetS on MSCs.

Background: Clonal hematopoiesis (CH) of indeterminate potential (CHIP) is a risk factor for cardiovascular disease. The relationship between CHIP and coronary microvascular dysfunction (CMD) is unknown. The current study examines the association between CHIP and CH with CMD and the potential relationships in risk for adverse cardiovascular outcomes. Methods: In this retrospective observational study, targeted next-generation sequencing was performed for 177 participants with no coronary artery disease who presented with chest pain and underwent routine coronary functional angiogram. Patients with somatic mutations in leukemia-associated driver genes in hematopoietic stem and progenitor cells were examined; CHIP was considered at a variant allele fraction ≥2%; CH was considered at a variant allele fraction ≥1%. CMD was defined as coronary flow reserve to intracoronary adenosine of ≤2. Major adverse cardiovascular events considered were myocardial infarction, coronary revascularization, or stroke. Results: A total of 177 participants were examined. Mean follow-up was 12±7 years. A total of 17 patients had CHIP and 28 had CH. Cases with CMD (n=19) were compared with controls with no CMD (n=158). Cases were 56±9 years, were 68% women, and had more CHIP (27%; P =0.028) and CH (42%; P =0.001) than controls. CMD was associated with independent risk for major adverse cardiovascular events (hazard ratio, 3.89 [95% CI, 1.21–12.56]; P =0.023), and 32% of this risk was mediated by CH. The risk mediated by CH was ≈0.5× as large as the direct effect of CMD on major adverse cardiovascular events. Conclusions: In humans, we observe patients with CMD are more likely to have CHIP, and nearly one-third of major adverse cardiovascular events in CMD are mediated by CH.

To address the ongoing shortage of organs available for replacement, xenotransplantation of hearts, corneas, skin, and kidneys has been attempted. However, a major obstacle facing xenotransplants is rejection due to a cycle of immune reactions to the graft. Both adaptive and innate immune systems contribute to this cycle, in which natural killer cells, macrophages, and T-cells play a significant role. While advancements in the field of genetic editing can circumvent some of these obstacles, biomarkers to identify and predict xenograft rejection remain to be standardized. Several T-cell markers, such as CD3, CD4, and CD8, are useful in both the diagnosis and prediction of xenograft rejection. Furthermore, an increase in the levels of various circulating DNA markers and microRNAs is also predictive of xenograft rejection. In this review, we summarize recent findings on the advancements in xenotransplantation, with a focus on pig-to-human, the role of immunity in xenograft rejection, and its biomarkers.

Autophagy eliminates excessive nutrients and maintains homeostasis. Obesity and metabolic syndrome (MetS) dysregulate autophagy, possibly partly due to mitochondria injury and inflammation. Elamipretide (ELAM) improves mitochondrial function. We hypothesized that MetS blunts kidney autophagy, which ELAM would restore. Domestic pigs were fed a control or MetS-inducing diet for 16 weeks. During the 4 last weeks, MetS pigs received subcutaneous injections of ELAM (0.1 mg/kg/day, MetS + ELAM) or vehicle (MetS), and kidneys were then harvested to measure protein expression of autophagy mediators and apoptosis. Systemic and renal venous levels of inflammatory cytokines were measured to calculate renal release. The function of isolated mitochondria was assessed by oxidative stress, energy production, and pro-apoptotic activity. MetS slightly downregulated renal expression of autophagy mediators including p62, ATG5-12, mTOR, and AMPK vs. control. Increased mitochondrial H2O2 production accompanied decreased ATP production, elevated apoptosis, and renal fibrosis. In MetS + ELAM, mito-protection restored autophagic protein expression, improved mitochondrial energetics, and blunted renal cytokine release and fibrosis. In vitro, mitoprotection restored mitochondrial membrane potential and reduced oxidative stress in injured proximal tubular epithelial cells. Our study suggests that swine MetS mildly affects renal autophagy, possibly secondary to mitochondrial damage, and may contribute to kidney structural damage in MetS.

Background: Uric acid (UA) concentration within carotid plaque and its association with cerebrovascular events have not been detected or quantified. Systemically, serum UA is a marker of inflammation and risk factor for atherosclerosis. However, its association with carotid plaque instability and stroke pathogenesis remains unclear. In patients undergoing carotid endarterectomy, we aimed to determine whether UA is present differentially in symptomatic versus asymptomatic carotid plaques and whether serum UA is associated with cerebrovascular symptoms (stroke, transient ischemic attack, or amaurosis fugax). Methods: Carotid atherosclerotic plaques were collected during carotid endarterectomy. The presence of UA was assessed using Gomori methenamine silver staining as well as anti-UA immunohistochemical staining and its quantity measured using an enzymatic colorimetric assay. Clinical information was obtained through a retrospective review of data. Results: UA was more commonly detected in symptomatic (n=23) compared with asymptomatic (n=9) carotid plaques by Gomori methenamine silver (20 [86.9%] versus 2 [22.2%]; P =0.001) and anti-UA immunohistochemistry (16 [69.5%] versus 1 [11.1%]; P =0.004). UA concentration was higher in symptomatic rather than asymptomatic plaques (25.1 [9.5] versus 17.9 [3.8] µg/g; P =0.021). Before carotid endarterectomy, serum UA levels were higher in symptomatic (n=341) compared with asymptomatic (n=146) patients (5.9 [interquartile range, 4.6–6.9] mg/dL versus 5.2 [interquartile range, 4.6–6.2] mg/dL; P =0.009). Conclusions: The current study supports a potential role of UA as a potential tissue participant and a systemic biomarker in the pathogenesis of carotid atherosclerosis. UA may provide a mechanistic explanation for plaque instability and subsequent ischemic cerebrovascular events.

Background: Scattered tubular-like cells (STCs) are dedifferentiated renal tubular cells endowed with progenitor-like characteristics to repair injured parenchymal cells. STCs may be damaged and rendered ineffective by renal artery stenosis (RAS), but the underlying processes remain unclear. We hypothesized that RAS alters the epigenetic landscape on DNA and the ensuing gene transcriptional profile of swine STCs. Methods: CD24+/CD133+ STCs were isolated from pig kidneys after 10 weeks of RAS or sham (n = 3 each) and their whole 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) profiles were examined by 5mC and 5hmC immunoprecipitation sequencing (MeDIP-/hMeDIP-seq, respectively). A subsequent integrated (MeDIP/hMeDIP-seq/mRNA-seq) analysis was performed by comparing all online available gene sets using Gene Set Enrichment Analysis. Apoptosis, proteolysis, and mitochondrial structure and function were subsequently evaluated in vitro. Results: Differential expression (DE) analysis revealed 239 genes with higher and 236 with lower 5mC levels and 275 genes with higher and 315 with lower 5hmC levels in RAS-STCs compared to Normal-STCs (fold change ≥1.4 or ≤0.7, p ≤ 0.05). Integrated MeDIP-/hMeDIP-seq/mRNA-seq analysis identified several overlapping (DE-5mC/mRNA and DE-5hmC/mRNA levels) genes primarily implicated in apoptosis, proteolysis, and mitochondrial functions. Furthermore, RAS-STCs exhibited decreased apoptosis, mitochondrial matrix density, and ATP production, and increased intracellular amino acid concentration and ubiquitin expression. Conclusions: Renal ischemia induces epigenetic changes in apoptosis-, proteolysis-, and mitochondria-related genes, which correlate with alterations in the transcriptomic profile and corresponding function of swine STCs. These observations may contribute to developing novel targeted interventions to preserve the reparative potency of STCs in renal disease.

ABSTRACT Background Renal artery stenosis (RAS) is an important cause of chronic kidney disease and secondary hypertension. In animal models, renal ischemia leads to downregulation of growth factor expression and loss of intrarenal microcirculation. However, little is known about the sequelae of large-vessel occlusive disease on the microcirculation within human kidneys. Method This study included five patients who underwent nephrectomy due to renovascular occlusion and seven nonstenotic discarded donor kidneys (four deceased donors). Micro-computed tomography was performed to assess microvascular spatial densities and tortuosity, an index of microvascular immaturity. Renal protein expression, gene expression and histology were studied in vitro using immunoblotting, polymerase chain reaction and staining. Results RAS demonstrated a loss of medium-sized vessels (0.2–0.3 mm) compared with donor kidneys (P = 0.037) and increased microvascular tortuosity. RAS kidneys had greater protein expression of angiopoietin-1, hypoxia-inducible factor-1α and thrombospondin-1 but lower protein expression of vascular endothelial growth factor (VEGF) than donor kidneys. Renal fibrosis, loss of peritubular capillaries (PTCs) and pericyte detachment were greater in RAS, yet they had more newly formed PTCs than donor kidneys. Therefore, our study quantified significant microvascular remodeling in the poststenotic human kidney. RAS induced renal microvascular loss, vascular remodeling and fibrosis. Despite downregulated VEGF, stenotic kidneys upregulated compensatory angiogenic pathways related to angiopoietin-1. Conclusions These observations underscore the nature of human RAS as a microvascular disease distal to main vessel stenosis and support therapeutic strategies directly targeting the poststenotic kidney microcirculation in patients with RAS.

Extracellular vesicles are important vectors for cell-cell communication and show potential value for diagnosis and treatment of kidney diseases. The pathologic diagnosis of kidney diseases relies on kidney biopsy, whereas collection of extracellular vesicles from urine or circulating blood may constitute a less invasive diagnostic tool. In particular, urinary extracellular vesicles released mainly from resident kidney cells might provide an alternative tool for detection of kidney injury. Because extracellular vesicles mirror many features of their parent cells, cargoes of several populations of urinary extracellular vesicles are promising biomarkers for disease processes, like diabetic kidney disease, kidney transplant, and lupus nephritis. Contrarily, extracellular vesicles derived from reparative cells, such as mesenchymal stem cells, tubular epithelial progenitor cells, and human umbilical cord blood represent promising regenerative tools for treatment of kidney diseases. Furthermore, induced pluripotent stem cells–derived and engineered extracellular vesicles are being developed for specific applications for the kidney. Nevertheless, some assumptions regarding the specificity and immunogenicity of extracellular vesicles remain to be established. This review focuses on the utility of extracellular vesicles as therapeutic and diagnostic (theranostic) tools in kidney diseases and future directions for studies.