Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular... Show morePreviously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate [eGFR] >30 mL/min/1.73 m(2)), and patients with DN (eGFR <30 mL/min/1.73 m(2)). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132). Show less
Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular... Show morePreviously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate [eGFR] >30 mL/min/1.73 m2), and patients with DN (eGFR <30 mL/min/1.73 m2). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132). Show less
Dysfunctional insulin secretion is a hallmark of type 2 diabetes (T2D). Interestingly, several islet microRNAs (miRNAs) are upregulated in T2D, including miR-132. We aimed to investigate whether in... Show moreDysfunctional insulin secretion is a hallmark of type 2 diabetes (T2D). Interestingly, several islet microRNAs (miRNAs) are upregulated in T2D, including miR-132. We aimed to investigate whether in vivo treatment with antagomir-132 lowers expression of miR-132 in islets thereby improving insulin secretion and lowering blood glucose. Mice injected with antagomir-132 for 24 h, had reduced expression of miR-132 expression in islets, decreased blood glucose, and increased insulin secretion. In isolated human islets treated with antagomir-132, insulin secretion from four of six donors increased. Target prediction coupled with analysis of miRNA-messenger RNA expression in human islets revealed DESI2, ARIH1, SLC25A28, DIAPH1, and FOXA1 to be targets of miR-132 that are conserved in both species. Increased expression of these targets was validated in mouse islets after antagomir-132 treatment. In conclusion, we identified a post-transcriptional role for miR-132 in insulin secretion, and demonstrated that systemic antagomir-132 treatment in mice can be used to improve insulin secretion and reduce blood glucose in vivo. Our study is a first step towards utilizing antagomirs as therapeutic agents to modulate islet miRNA levels to improve beta cell function. Show less
There is an increasing prevalence of chronic kidney disease (CKD), which associates with the development of interstitial fibrosis. Pericytes (perivascular fibroblasts) provide a major source of... Show moreThere is an increasing prevalence of chronic kidney disease (CKD), which associates with the development of interstitial fibrosis. Pericytes (perivascular fibroblasts) provide a major source of alpha-SMA-positive myofibroblasts that are responsible for the excessive deposition of extracellular matrix. In order to identify pericyte long non-coding RNAs (lncRNAs) that could serve as a target to decrease myofibroblast formation and counteract the progression of kidney fibrosis we employed two models of experimental kidney injury, one focused on kidney fibrosis (unilateral ureteral obstruction; UUO), and one focused on acute kidney injury that yields kidney fibrosis in the longer term (unilateral ischemia-reperfusion injury; IRI). This was performed in FoxD1-GC;tdTomato stromal cell reporter mice that allowed pericyte fate tracing. Tomato red-positive FoxD1-derivative cells of control and injured kidneys were FACS-sorted and used for lncRNA and mRNA profiling yielding a distinctive transcriptional signature of pericytes and myofibroblasts with 244 and 586 differentially expressed lncRNAs (> twofold, P < 0.05), in the UUO and IRI models, respectively. Next, we selected two differentially expressed and conserved lncRNAs, Rian (RNA imprinted and accumulated in nucleus) and Miat (Myocardial infarction associated transcript), and explored their potential regulatory role in myofibroblast formation through knockdown of their function with gapmers. While Miat was upregulated in myofibroblasts of UUO and IRI in mice, gapmer silencing of Miat attenuated myofibroblast formation as evidenced by decreased expression of alpha-SMA, col1 alpha 1, SMAD2, and SMAD3, as well as decreased alpha-SMA and pro-collagen-1 alpha 1 protein levels. In contrast, silencing Rian, which was found to be downregulated in kidney myofibroblast after IRI and UUO, resulted in increased myofibroblast formation. In addition, we found microRNAs that were previously linked to Miat (miR-150) and Rian (14q32 miRNA cluster), to be dysregulated in the FoxD1-derivative cells, suggesting a possible interaction between miRNAs and these lncRNAs in myofibroblast formation. Taken together, lncRNAs play a regulatory role in myofibroblast formation, possibly through interacting with miRNA regulation, implicating that understanding their biology and their modulation may have the potential to counteract the development of renal fibrosis. Show less
Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides... Show moreKidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor beta (TGF-beta) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n= 10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (similar to 55% reduction, P< 0.05) and alpha-smooth muscle actin (alpha-SMA; similar to 40% reduction, P< 0.05). Moreover, the number of alpha-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation. Show less
Bijkerk, R.; Trimpert, C.; Solingen, C. van; Bruin, R.G. de; Florijn, B.W.; Kooijman, S.; ... ; Zonneveld, A.J. van 2018
CVD is the primary cause of death among men and women worldwide. Nevertheless, our comprehension of how CVD progresses in women and elicits clinical outcomes is lacking, leading CVD to be under... Show moreCVD is the primary cause of death among men and women worldwide. Nevertheless, our comprehension of how CVD progresses in women and elicits clinical outcomes is lacking, leading CVD to be under-diagnosed and under-treated in women. A clear example of this differential presentation of CVD pathophysiologies in females is the strikingly higher prevalence of heart failure with preserved ejection fraction (HFpEF). Women with a history of preeclampsia, or those that present co-morbidities such as obesity, hypertension and diabetes mellitus are at increased risk to develop HFpEF. Long understood to be a critical CVD risk factor, our understanding of how gender differentially impacts the development of CVD has been greatly expanded by extensive genomic and transcriptomic studies. These studies uncovered a pivotal role for differential miRNA expression in response to systemic inflammation, where their coordinated expression forms a post-transcriptional regulatory network that instigates microcirculation defects. Importantly, the potential sex-biased expression of given miRNAs may explain sex-specific cardiovascular pathophysiologies in women, such as HFpEF. Sex-biased miRNAs are regulated by estrogen (E2) in their transcription and processing or are expressed from loci on the X-chromosome due to incomplete X-chromosome inactivation. Interestingly, while E2-induced miRNAs predominantly appear to serve protective functions, it could be argued that many X-linked miRNAs have been found to challenge microvascular and myocardial integrity. Therefore, menopausal E2 deficiency, resulting in protective miRNA loss, and the augmentation of X-linked miRNA expression, may well contribute to the molecular mechanisms that underlie the female-specific cardiovascular aetiology in HFpEF. Show less
Pelzer, N.; Bijkerk, R.; Reinders, M.; Zonneveld, A.J. van; Ferrari, M.; Maagdenberg, A. van den; ... ; Terwindt, G. 2017
Conclusions/interpretation Taken together, circulating MBL levels are associated with diabetic nephropathy and are dependent on glycaemic control, possibly in an MBL2-genotype-dependent manner.
