Stem cell-derived extracellular vesicles for renal repair: do cardiovascular comorbidities matter?

Abstract

Extracellular vesicle (EV)-based regenerative therapy has shown promising results in preclinical models of renal disease and might be useful for patients with several forms of chronic kidney disease. However, individuals with chronic kidney disease often present with comorbidities, including obesity, hypertension, diabetes, or even metabolic syndrome, which may alter the endogenous characteristics and impair the reparative capacity of stem cells and their daughter EVs. This brief review summarizes evidence of alterations in the morphology, cargo, and function of mesenchymal stem cells and mesenchymal stem cell-derived EVs in the face of cardiovascular disease. We further discuss the important ramifications for their use in patients with kidney disease.

INTRODUCTION

Regenerative strategies are leaving an important mark on several areas of medicine, including nephrology. Developing novel therapies to attenuate the progression of renal failure represents a major goal for clinicians treating patients with chronic kidney disease (CKD), a condition that affects nearly 15% of the United States population and over 200 million people worldwide (53). Mesenchymal stem cells (MSCs) are undifferentiated nonembryonic stem cells present in adult tissues that possess important proangiogenic and immunomodulatory properties and can differentiate into a broad spectrum of cell lineages (3). Moreover, these cells can be isolated from a myriad of tissues, including adipose tissue and bone marrow, which makes them ideal candidates for regenerative therapy (36). Available experimental evidence demonstrates that MSCs contribute to cellular repair and ameliorate renal injury in many forms of CKD (11), prompting several clinical trials testing their safety and efficacy in patients with renal disease (35). The primary mechanism of action of MSCs is the paracrine release of growth factors and extracellular vesicles (EVs), membrane microparticles that shuttle genetic and protein information to activate a repair program in recipient cells (51). Recent studies have shown that delivery of MSC-derived EVs improves renal outcomes in several animal models of CKD (1). However, patients with CKD often present with comorbidities, which may alter the endogenous characteristics and EV cargo of MSCs, limiting their potency and autologous therapeutic potential in these individuals. Here, we summarize studies that explored the impact of cardiovascular risk factors on the morphology, cargo, and function of MSC-derived EVs and discuss the clinical implications of these findings (Fig. 1 and Table 1).

Fig. 1.

Cardiovascular risk factors may interfere with the endogenous characteristics and extracellular vesicle (EV) cargo of mesenchymal stem cells (MSCs). Obesity, hypertension, insulin resistance, and hyperlipidemia may alter the mRNA, microRNA, and protein profile of MSCs and induce changes in the morphology and cargo of MSC-derived EVs.

Table 1.

Experimental studies testing the effects of cardiovascular comorbidities on the reparative capacity of stem cells and stem cell-derived EVs

Species	Sex	Model	Number of subjects	Duration	Source of Stem Cells	Main findings	Reference
Pig	Female	Diet-induced MetS	12	16 wk	Adipose tissue	MetS alters expression of insulin signaling-related genes	9
Rats	Male	Cholesterol loading	6	2 mo	Bone marrow	Cholesterol loading retards cellular senescence of MSCs	61
Pig	Female	Diet-induced MetS	12	16 wk	Adipose tissue	MetS induces release of smaller EVs from MSCs	8
Mice	Female	Cholesterol loading	8	6 wk	Bone marrow	Cholesterol loading affects osteoblastic differentiation of MSCs	33
Pig	Female	Diet-induced MetS	12	16 wk	Adipose tissue	MetS induces changes in the expression of senescence-related microRNAs that regulate MSC senescence	42
Pig	Female	Diet-induced MetS	10	16 wk	Adipose tissue	MetS interferes with mitochondrial protein import by modulating the expression of genes encoding for transporters of mitochondrial proteins	2
Pig	Female	Diet-induced MetS	14	16 wk	Adipose tissue	MetS enhances MSC adipogenic and osteogenic differentiation, inflammation, and senescence	63
Pig	Female	Diet-induced MetS	8	16 wk	Adipose tissue	MetS alters the genetic and protein content of MSCs	48
Pig	Female	Diet-induced MetS	10	16 wk	Adipose tissue	MetS Interferes with packaging of proteins within MSC-derived EVs	18
Pig	Female	Diet-induced MetS	14	16 wk	Adipose tissue	MetS impairs mitochondrial structure and function partly through miRNA-induced mitochondrial gene regulation	41

EVs, extracellular vesicles; MSCs, mesenchymal stem cells; MetS, metabolic syndrome.

MSCs AND MSC-DERIVED EVs FOR RENAL REPAIR

There are currently dozens of clinical trials worldwide testing the safety and efficacy of MSCs to treat patients with different forms of renal disease (50), some of which have shown auspicious results in terms of recovery of kidney function. For example, a multicenter, randomized, double-blind, placebo-controlled trial has shown that a single intravenous infusion of allogeneic bone marrow-derived MSCs improved estimated glomerular filtration rate after 12 wk in patients with diabetic nephropathy (47). Similarly, intra-arterial delivery of autologous MSCs in patients with renovascular disease increases poststenotic kidney renal blood flow and reduces renal tissue hypoxia, assessed by multidetector computed tomography (MDCT) and blood oxygen level-dependent MRI, respectively (52). In addition, experimental data have shown that MSC-derived EV therapy improves renal outcomes in several models of CKD (1, 45), suggesting that their delivery may be an attractive cell-free therapy for renal disease. For example, we have recently shown that intra-renal delivery of MSC-derived EVs attenuated tissue inflammation and microvascular loss and, in turn, improved stenotic kidney hemodynamics and function (assessed by MDCT) in female pigs with chronic renovascular disease (13, 15). In line with this, a recent clinical trial demonstrated that delivery of MSC-derived EV therapy was safe, ameliorated renal inflammation, and improved estimated glomerular filtration rate, serum creatinine, blood urea, and the urinary albumin-to-creatinine ratio in patients with CKD (46). These particles offer some exciting advantages over MSCs. Freezing, thawing, and storage conditions are less critical for EVs than for MSCs. Furthermore, they can much easier be produced in a scaled manner than cellular therapeutics and do not self-replicate, which minimizes the endogenous tumor formation potential (24). Interestingly, studies in a 5/6 subtotal nephrectomy mouse model have shown that MSCs and MSC-derived EVs have similar potential to attenuate tissue injury (tubulointerstitial fibrosis, lymphocyte infiltrates, and tubular atrophy) and preserve kidney function (blood urea nitrogen, serum creatinine, uric acid, and proteinuria) (26). EVs recapitulate the beneficial effects in kidney repair of MSCs and may also confer additional renoprotective effects (25). While future long-term followup clinical studies are needed to confirm the persistence of these beneficial effects, these observations position MSC-derived EVs as a realistic clinical tool to treat patients with CKD.

CARDIOVASCULAR COMORBIDITIES AND MSCs

Metabolic syndrome (MetS) is a prevalent condition that affects over 20% of adults in Western populations (4) and is associated with the development and progression of CKD (59). According to Adult Treatment Panel-III guidelines, MetS is defined by the presence of three or more of the following: fasting plasma glucose ≥ 110 mg/dL, serum triglycerides ≥ 150 mg/dL, serum HDL-cholesterol < 40 mg/dL, blood pressure ≥ 130/85 mmHg or on antihypertensive medication, or waist girth > 102 cm (19). MetS and its components can foster chronic inflammation, oxidative stress, endothelial dysfunction, and activation of the renin-angiotensin-aldosterone system, promoting the progression of kidney cell damage (62). Adipose tissue is an increasingly popular source of MSCs for clinical applications (57). However, the components of MetS can induce microenvironmental changes in adipose tissue, including inflammation (22), endoplasmic reticulum stress (29), alterations in insulin (27) and angiotensin II signaling (31), mitochondrial dysfunction (32), and cellular senescence (44). MetS-induced oxidative stress can also promote insulin resistance of adipocytes, increasing secretion of leptin and proinflammatory cytokines (38). Hyperglycemia may further aggravate adipose cell dysfunction, contributing to inflammation and insulin resistance (54). Therefore, the efficacy of MSCs immersed in the noxious microenvironment of MetS may be compromised for autologous delivery, the preferred type of MSC transplant for clinical trials. In recent years, we have explored the potential deleterious effects of MetS on MSCs using a novel swine model of diet-induced MetS that recapitulates the main features of the human disease (49). We comprehensively characterized the transcriptomic and proteomic profile of adipose tissue-derived MSCs using high-throughput RNA sequencing and mass spectrometry proteomic analysis (48). Our studies revealed that MetS altered the content of genes, proteins, and microRNAs of swine MSCs, in particular those involved in insulin signaling (9), senescence (42), and mitochondrial function (2, 41). Functionally, we found that although MSC proliferation and migration remained unaltered, MetS enhanced adipogenic and osteogenic differentiation, inflammation, and senescence, but impaired mitochondrial respiratory function (48, 63). Notably, MetS-induced changes in MSC function are partly mediated by TNF-α, a proinflammatory cytokine highly expressed in adipose tissue (28). Therefore, MetS-induced changes in adipose tissue may trigger alterations in the gene, protein, and microRNA profile of MSCs, contributing to cellular activation and adaptive responses.

Each of the components of MetS may account for its effects on MSCs, given that hyperlipidemia, hypertension, obesity, and diabetes can also individually modulate MSC function. Cholesterol loading stimulates osteoblastic differentiation (33) and may retard senescence (61) of bone marrow-derived MSCs. Hypertension can also alter the number and function of both progenitor and stem cells. Circulating endothelial progenitor cells are immature endothelial cells mobilized endogenously from the bone marrow in response to ischemia (6). These cells express the cell surface glycoprotein CD34 and the kinase insert domain-containing receptor (KDR) and play a central role in vascular homeostasis by promoting neovascularization and endothelial repair (5, 14). However, the in vivo endothelial repair capacity of early endothelial progenitor cells is reduced in patients with prehypertension and hypertension (23). Similarly, the number of systemic circulating CD34⁺/kinase insert domain-containing receptor⁺ endothelial progenitor cells is decreased in renovascular disease and essential hypertensive patients compared with healthy volunteers (10). Contrarily, patients with essential hypertension have increased circulating MSCs compared with normotensive patients, and their number is correlated with the degree of left ventricular hypertrophy (37), implying that these cells may participate in cardiac remodeling. MSCs of patients with type 2 diabetes have increased apoptosis and limited multipotency (58) as well as altered expression of surface antigens, morphology, cytokine secretion, proliferation, and differentiation (21, 30). Finally, obesity can also modulate the paracrine function of MSCs (60).

Cardiovascular diseases are recognized age-related conditions. Aging can cause changes in the heart and blood vessels, representing an important risk factor for developing cardiovascular disease (55). Likewise, aging can compromise the functional characteristics of MSCs. Adipose tissue-derived MSCs isolated from elder individuals exhibit senescent features, apoptosis, and decreased viability (7). Similarly, MSC numbers obtained by bone marrow aspiration decline with donor age (56). Therefore, the therapeutic application of MSCs may be limited in these patients.

CARDIOVASCULAR RISK FACTORS AND EVs

Far from being a random process, the genetic and protein content of MSC-derived EVs is meticulously selected for packaging. Genes, proteins, and microRNAs are either enriched or selectively excluded from EVs, resulting in a versatile cargo capable of modulating diverse cellular functions of the target cells (12, 16, 17). Consequently, MetS-induced changes in the content and function of adipose tissue-derived MSCs may also impact on their EV progeny. Recently, we reported that swine MetS altered the size distribution of MSC-derived EVs compared with those obtained from lean pigs toward smaller vesicles packed with genes commonly associated with exosomes (8), although the overall EV number released by MetS MSCs remained unchanged. MetS also modulates the mRNA cargo packed within porcine adipose tissue MSC-derived EVs, which contain genes involved in translational regulation and modulation of inflammation (39). Notably, coculture with MetS EVs increases renal tubular cell inflammation in vitro, suggesting that MetS may affect the immunomodulatory function of MSCs by modifying mRNA profiles of their EVs. Similarly, MetS alters the microRNA content of EVs, selectively packing microRNAs that modulate pathways involved in the development of MetS and its complications (40), and modifies the protein cargo of MSC-derived EVs, favoring the inclusion of proteins linked to several proinflammatory pathways (18). Notably, treatment with MetS EVs increases expression of proinflammatory cytokines and fosters NF-κB activation in tubular cells in vitro, implying that the proinflammatory cargo of EVs may impair the paracrine immunomodulatory potential of MSCs. In line with this, EVs released from adipose tissue-derived stem cells recovered from obese patients show impaired angiogenic potential compared with those obtained from nonobese individuals (60). However, in the context of type 1 diabetes, MSC-released EVs retain their biological effect (20), implying that cardiovascular risk factors might have distinct effect on EV function. Altogether, these observations provide insights into the associated mechanisms leading to EV release and suggest that cardiovascular risk factors may impair the ability of MSCs to repair damaged tissues.

PERSPECTIVES

Cardiovascular risk factor-induced changes in the endogenous characteristics and function of MSCs and MSC-derived EVs may diminish the regenerative benefits of autologous transplantation and impact on different aspects of their autologous clinical application. For example, MetS-induced changes in the content or size distribution of EVs may require adjustments in the optimal dose regimen. Likewise, preconditioning strategies (e.g., hypoxia, insulin-like growth factor-1, and melatonin) (34, 43) may enhance the survival and potency of MSCs to overcome changes in the packaging cargo of MSC-derived EVs. Finally, pretreatment with anti-inflammatory strategies may be useful to blunt their transdifferentiation into adipocytes and prevent some of the functional abnormalities of diabetic and MetS MSCs. Additional research is warranted to explore if cardiovascular disease induces epigenetic or posttranscriptional alterations on MSC function to interfere with the formation, release, and cargo of their daughter EVs. Further studies are also needed to test the efficacy of MSC-derived EVs in vivo and explore whether diabetic and MetS MSCs and EVs differ from those obtained from healthy individuals.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-106427, DK-122137, DK-104273, DK-120292, and DK-102325.

DISCLOSURES

L. O. Lerman received grant funding from Novo Nordisk and is an advisor to Weijian Technologies. No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

A.E. prepared figures; A.E. drafted manuscript; L.O.L. edited and revised manuscript; A.E. and L.O.L. approved final version of manuscript.