No abstract

Vascular progenitor cells show promise for the treatment of microvasculature endothelial injury. We investigated the function of renal artery progenitor cells derived from radical nephrectomy patients, in animal models of acute ischemic and hyperperfusion injuries. Present in human adventitia, CD34positive/CD105negative cells were clonal and expressed transcription factors Sox2/Oct4 as well as surface markers CXCR4 (CD184)/KDR(CD309) consistent with endothelial progenitor cells. Termed renal artery-derived vascular progenitor cells (RAPC), injected cells were associated with decreased serum creatinine after ischemia/reperfusion, reduced albuminuria after hyperperfusion, and improved blood flow in both models. A small population of RAPC integrated with the renal microvasculature following either experimental injury. At a cellular level, RAPC promoted local endothelial migration in co-culture. Profiling of RAPC microRNA identified high levels of miRNA 218; also found at high levels in exosomes isolated from RAPC conditioned media after cell contact for 24 hours. After hydrogen peroxide-induced endothelial injury, RAPC exosomes harbored Robo-1 transcript; a gene known to be regulated by mir218. Such exosomes enhanced endothelial cell migration in culture in the absence of RAPC. Thus, our work shows the feasibility of pre-emptive pro-angiogenic progenitor cell procurement from a targeted patient population and potential therapeutic use in the form of autologous cell transplantation.

Park et al describe a novel KLF4-mediated pathway that promotes chromic myeloid leukemia (CML) stem cell (LSC) survival. Deletion of KLF4 in a mouse model of CML decreases LSC survival through repression of Dyrk2, resulting in c-Myc depletion and increased p53 activity.

These results suggest GPx3 deficiency is a substantive contributing factor to the development of kidney disease-induced cardiac disease.

Background The sodium channel, Na v 1.5, encoded by SCN 5A , undergoes developmentally regulated splicing from inclusion of exon 6A in the fetal heart to exon 6B in adults. These mutually exclusive exons differ in 7 amino acids altering the electrophysiological properties of the Na v 1.5 channel. In myotonic dystrophy type 1, SCN 5A is mis‐spliced such that the fetal pattern of exon 6A inclusion is detected in adult hearts. Cardiac manifestations of myotonic dystrophy type 1 include conduction defects and arrhythmias and are the second‐leading cause of death. Methods and Results This work aimed to determine the impact of SCN 5A mis‐splicing on cardiac function. We used clustered regularly interspaced short palindromic repeat ( CRISPR) /CRISPR‐associated protein 9 (Cas9) to delete Scn5a exon 6B in mice, thereby redirecting splicing toward exon 6A. These mice exhibit prolonged PR and QRS intervals, slowed conduction velocity, extended action potential duration, and are highly susceptible to arrhythmias. Conclusions Our findings highlight a nonmutational pathological mechanism of arrhythmias and conduction defects as a result of mis‐splicing of the predominant cardiac sodium channel. Animals homozygous for the deleted exon express only the fetal isoform and have more‐severe phenotypes than heterozygotes that also express the adult isoform. This observation is directly relevant to myotonic dystrophy type 1, and possibly pathological arrhythmias, in which individuals differ with regard to the ratios of the isoforms expressed.

Ischemia due to hypoperfusion is one of the most common forms of acute kidney injury. We hypothesized that kidney hypoxia initiates the up-regulation of miR-218 expression in endothelial progenitor cells (EPCs) to guide endocapillary repair. Murine renal arteryederived EPCs (CD34 þ /CD105 À ) showed down-regulation of mmu-Mir218-5p/U6 RNA ratio after ischemic injury, while in human renal arteries, MIR218-5p expression was up-regulated after ischemic injury. MIR218 expression was clarified in cell culture experiments in which increases in both SLIT3 and MIR218-2-5p expressions were observed after 5 minutes of hypoxia. ROBO1 transcript, a downstream target of MIR218-2-5p, showed inverse expression to MIR218-2-5p. EPCs transfected with a MIR218-5p inhibitor in three-dimensional normoxic culture showed premature capillary formation. Organized progenitor cell movement was reconstituted when cells were co-transfected with Dicer siRNA and low-dose Mir218-5p mimic. A Mir218-2 knockout was generated to assess the significance of miR-218-2 in a mammalian model. Mir218-2-5p expression was decreased in Mir218-2 À/À embryos at E16.5. Mir218-2 À/À decreased CD34 þ angioblasts in the ureteric bud at E16.5 and were nonviable. Mir218-2 þ/À decreased peritubular capillary density at postnatal day 14 and increased serum creatinine after ischemia in adult mice. Systemic injection of miR-218-5p decreased serum creatinine after injury. These experiments demonstrate that miR-218 expression can be triggered by hypoxia and modulates EPC migration in the kidney.

Vascular inflammation is a major contributor to the severity of acute kidney injury. In the context of vasospasm-independent reperfusion injury we studied the potential anti-inflammatory role of the Gα-related RGS protein, RGS4. Transgenic RGS4 mice were resistant to 25 minute injury, although post-ischemic renal arteriolar diameter was equal to the wild type early after injury. A 10 minute unilateral injury was performed to study reperfusion without vasospasm. Eighteen hours after injury blood flow was decreased in the inner cortex of wild type mice with preservation of tubular architecture. Angiotensin II levels in the kidneys of wild type and transgenic mice were elevated in a sub-vasoconstrictive range 12 and 18 hours after injury. Angiotensin II stimulated pre-glomerular vascular smooth muscle cells (VSMC) to secrete the macrophage chemoattractant, RANTES; a process decreased by angiotensin II R2 (AT2) inhibition. However, RANTES increased when RGS4 expression was suppressed implicating Gα protein activation in an AT2-RGS4-dependent pathway. RGS4 function, specific to VSMC, was tested in a conditional VSMC-specific RGS4 knockout showing high macrophage density by T2 MRI compared to transgenic and non-transgenic mice after the 10 minute injury. Arteriolar diameter of this knockout was unchanged at successive time points after injury. Thus, RGS4 expression, specific to renal VSMC, inhibits angiotensin II-mediated cytokine signaling and macrophage recruitment during reperfusion, distinct from vasomotor regulation.

Summary Multiple strategies have been developed to efficiently differentiate human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we describe a protocol for measuring three key functional parameters of hiPSC-CMs, including contractile function, calcium (Ca 2+ ) handling, and action potential. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021) .