Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis.

To investigate the contribution of ion channels to ciliogenesis, we carried out a small interfering RNA (siRNA)-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles regulate renal ciliogenesis; KCNQ1-p.R518X, -p.A178T and -p.K362R could not rescue ciliogenesis after Kcnq1-siRNA-mediated depletion in contrast to wild-type KCNQ1 and benign KCNQ1-p.R518Q, suggesting that the ion channel function of KCNQ1 regulates ciliogenesis. In contrast, we demonstrate that the ion channel function of KCNJ10 is independent of its effect on ciliogenesis. Our data suggest that these four ion channels regulate renal ciliogenesis through the periciliary diffusion barrier or the ciliary pocket, with potential implication as genetic contributors to ciliopathy pathophysiology. The new functional roles of a subset of ion channels provide new insights into the disease pathogenesis of channelopathies, which might suggest future therapeutic approaches.

Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells.

Signaling between endothelial cells, endothelial progenitor cells, and stromal cells is crucial for the establishment and maintenance of vascular integrity and involves exosomes, among other signaling pathways. Exosomes are important mediators of intercellular communication in immune signaling, tumor survival, stress responses, and angiogenesis. The ability of exosomes to incorporate and transfer messenger RNAs (mRNAs) encoding for "acquired" proteins or micro RNAs (miRNAs) repressing "resident" mRNA translation suggests that they can influence the physiological behavior of recipient cells. We demonstrate that miR-214, an miRNA that controls endothelial cell function and angiogenesis, plays a dominant role in exosome-mediated signaling between endothelial cells. Endothelial cell-derived exosomes stimulated migration and angiogenesis in recipient cells, whereas exosomes from miR-214-depleted endothelial cells failed to stimulate these processes. Exosomes containing miR-214 repressed the expression of ataxia telangiectasia mutated in recipient cells, thereby preventing senescence and allowing blood vessel formation. Concordantly, specific reduction of miR-214 content in exosome-producing endothelial cells abolishes the angiogenesis stimulatory function of the resulting exosomes. Collectively, our data indicate that endothelial cells release miR-214-containing exosomes to stimulate angiogenesis through the silencing of ataxia telangiectasia mutated in neighboring target cells.

Neovascularization capacity of mesenchymal stromal cells from critical limb ischemia patients is equivalent to healthy controls.

Critical limb ischemia (CLI) is often poorly treatable by conventional management and alternatives such as autologous cell therapy are increasingly investigated. Whereas previous studies showed a substantial impairment of neovascularization capacity in primary bone-marrow (BM) isolates from patients, little is known about dysfunction in patient-derived BM mesenchymal stromal cells (MSCs). In this study, we have compared CLI-MSCs to healthy controls using gene expression profiling and functional assays for differentiation, senescence and in vitro and in vivo pro-angiogenic ability. Whereas no differentially expressed genes were found and adipogenic and osteogenic differentiation did not significantly differ between groups, chondrogenic differentiation was impaired in CLI-MSCs, potentially as a consequence of increased senescence. Migration experiments showed no differences in growth factor sensitivity and secretion between CLI- and control MSCs. In a murine hind-limb ischemia model, recovery of perfusion was enhanced in MSC-treated mice compared to vehicle controls (71 ± 24% versus 44 ± 11%; P < 1 × 10(-6)). CLI-MSC- and control-MSC-treated animals showed nearly identical amounts of reperfusion (ratio CLI:Control = 0.98, 95% CI = 0.82-1.14), meeting our criteria for statistical equivalence. The neovascularization capacity of MSCs derived from CLI-patients is not compromised and equivalent to that of control MSCs, suggesting that autologous MSCs are suitable for cell therapy in CLI patients.

Off-the-Shelf Synthetic Biodegradable Grafts Transform In Situ into a Living Arteriovenous Fistula in a Large Animal Model.

Current vascular access options require frequent interventions. In situ tissue engineering (TE) may overcome these limitations by combining the initial success of synthetic grafts with long-term advantages of autologous vessels by using biodegradable grafts that transform into autologous vascular tissue at the site of implantation. Scaffolds (6 mm-Ø) made of supramolecular polycarbonate-bisurea (PC-BU), with a polycaprolactone (PCL) anti-kinking-coil, are implanted between the carotid artery and jugular vein in goats. A subset is bio-functionalized using bisurea-modified-Stromal cell-derived factor-1α (SDF1α) derived peptides and ePTFE grafts as controls. Grafts are explanted after 1 and 3 months, and evaluated for material degradation, tissue formation, compliance, and patency. At 3 months, the scaffold is resorbed and replaced by vascular neo-tissue, including elastin, contractile markers, and endothelial lining. No dilations, ruptures, or aneurysms are observed and grafts are successfully cannulated at termination. SDF-1α-peptide-biofunctionalization does not influence outcomes. Patency is lower in TE grafts (50%) compared to controls (100% patency), predominantly caused by intimal hyperplasia. Rapid remodeling of a synthetic, biodegradable vascular scaffold into a living, compliant arteriovenous fistula is demonstrated in a large animal model. Despite lower patency compared to ePTFE, transformation into autologous and compliant living tissue with self-healing capacity may have long-term advantages.

Lack of haptoglobin results in unbalanced VEGFα/angiopoietin-1 expression, intramural hemorrhage and impaired wound healing after myocardial infarction.

Decreased haptoglobin (Hp) functionality due to allelic variations is associated with worsened outcome in patients after myocardial infarction (MI). However, mechanisms through which haptoglobin deficiency impairs cardiac repair remain to be elucidated. In the present study, we identified novel molecular alterations mediated by Hp involved in early and late cardiac repair responses after left coronary artery ligation in Hp(-/-) and wild-type (WT) mice. We observed a higher mortality rate in Hp(-/-) mice despite similar infarct size between groups. Deaths were commonly caused by cardiac rupture in Hp(-/-) animals. Histological analysis of 3 and 7days old non-ruptured infarcted hearts revealed more frequent and more severe intramural hemorrhage and increased leukocyte infiltration in Hp(-/-) mice. Analyses of non-ruptured hearts revealed increased oxidative stress, reduced PAI-1 activity and enhanced VEGFα transcription in Hp(-/-) mice. In line with these observations, we found increased microvascular permeability in Hp(-/-) hearts 3days after infarction. In vitro, haptoglobin prevented hemoglobin-induced oxidative stress and restored VEGF/Ang-1 balance in endothelial cell cultures. During long-term follow-up of the surviving animals, we observed altered matrix turnover, impaired scar formation and worsened cardiac function and geometry in Hp(-/-)mice. In conclusion, haptoglobin deficiency severely deteriorates tissue repair and cardiac performance after experimental MI. Haptoglobin plays a crucial role in both short- and long-term cardiac repair responses by reducing oxidative stress, maintaining microvascular integrity, myocardial architecture and proper scar formation.

The effect of chronic kidney disease on tissue formation of in situ tissue-engineered vascular grafts.

Vascular in situ tissue engineering encompasses a single-step approach with a wide adaptive potential and true off-the-shelf availability for vascular grafts. However, a synchronized balance between breakdown of the scaffold material and neo-tissue formation is essential. Chronic kidney disease (CKD) may influence this balance, lowering the usability of these grafts for vascular access in end-stage CKD patients on dialysis. We aimed to investigate the effects of CKD on in vivo scaffold breakdown and tissue formation in grafts made of electrospun, modular, supramolecular polycarbonate with ureido-pyrimidinone moieties (PC-UPy). We implanted PC-UPy aortic interposition grafts (n = 40) in a rat 5/6th nephrectomy model that mimics systemic conditions in human CKD patients. We studied patency, mechanical stability, extracellular matrix (ECM) components, total cellularity, vascular tissue formation, and vascular calcification in CKD and healthy rats at 2, 4, 8, and 12 weeks post-implantation. Our study shows successful in vivo application of a slow-degrading small-diameter vascular graft that supports adequate in situ vascular tissue formation. Despite systemic inflammation associated with CKD, no influence of CKD on patency (Sham: 95% vs CKD: 100%), mechanical stability, ECM formation (Sirius red+, Sham 16.5% vs CKD 25.0%-p:0.83), tissue composition, and immune cell infiltration was found. We did find a limited increase in vascular calcification at 12 weeks (Sham 0.08% vs CKD 0.80%-p:0.02) in grafts implanted in CKD animals. However, this was not associated with increased stiffness in the explants. Our findings suggest that disease-specific graft design may not be necessary for use in CKD patients on dialysis.

Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia-reperfusion in vivo.

Accumulating evidence indicates that programmed necrosis plays a critical role in cell death during ischemia-reperfusion. Necrostatin-1 (Nec-1), a small molecule capable of inhibiting a key regulator of programmed necrosis (RIP1), was shown to prevent necrotic cell death in experimental models including cardiac ischemia. However, no functional follow-up was performed and the action of Nec-1 remains unclear. Here, we studied whether Nec-1 inhibits RIP1-dependent necrosis and leads to long-term improvements after ischemia-reperfusion in vivo. Mice underwent 30 min of ischemia and received, 5 min before reperfusion, 3.3 mg/kg Nec-1 or vehicle treatment, followed by reperfusion. Nec-1 administration reduced infarct size to 26.3 ± 1.3% (P = 0.001) compared to 38.6 ± 1.7% in vehicle-treated animals. Furthermore, Nec-1 inhibited RIP1/RIP3 phosphorylation in vivo and significantly reduced necrotic cell death, while apoptotic cell death remained constant. By using MRI, cardiac dimensions and function were assessed before and 28 days after surgery. Nec-1-treated mice displayed less adverse remodeling (end-diastolic volume 63.5 ± 2.8 vs. 74.9 ± 2.8 µl, P = 0.031) and preserved cardiac performance (ejection fraction 45.81 ± 2.05 vs. 36.03 ± 2.37%, P = 0.016). Nec-1 treatment significantly reduced inflammatory influx, tumor necrosis factor-α mRNA levels and oxidative stress levels. Interestingly, this was accompanied by significant changes in the expression signature of oxidative stress genes. Administration of Nec-1 at the onset of reperfusion inhibits RIP1-dependent necrosis in vivo, leading to infarct size reduction and preservation of cardiac function. The cardioprotective effect of Nec-1 highlights the importance of necrotic cell death in the ischemic heart, thereby opening a new direction for therapy in patients with myocardial infarction.

Human Bone Marrow Mononuclear Cells Do Not Improve Limb Perfusion in the Hindlimb Ischemia Model.

Effective treatments for chronic limb-threatening ischemia are lacking. (Pre)clinical studies on administration of bone marrow (BM) mononuclear cells (MNCs) and BM-derived mesenchymal stromal cells (MSCs) have shown variable results and no studies have directly compared administration of human BM MNCs and BM MSCs in in vivo models. We studied the effect of intramuscular administration of human BM-derived MNCs and MSCs on limb perfusion in the murine hindlimb ischemia (HLI) model. Human BM MNCs and MSCs were obtained from healthy consenting donors. Both cell types were cryopreserved before use. Twenty-four hours after induction of HLI, nude NMRI mice were randomized to receive intramuscular administration of human BM MNCs (n = 13), or BM MSCs (n = 14), or vehicle control (n = 19) in various doses. Limb perfusion was measured using laser Doppler imaging on day 0, 1, 4, 7, 10, and 14. Intramuscular injection of human BM MNCs did not improve limb perfusion as compared with vehicle over the 2 weeks after cell administration (P = 0.88, mean relative perfusion for vehicle 0.56 ± 0.04 and 0.53 ± 0.04 for BM MNCs at day 14). Administration of human BM MSCs significantly improved limb perfusion as compared with both BM MNCs and vehicle (P ≤ 0.001, mean relative perfusion at day 14 0.79 ± 0.06). Our data suggest that BM MNCs are less suitable than BM MSCs for cell-based therapy that aims to restore perfusion.

Evaluation of infarcted murine heart function: comparison of prospectively triggered with self-gated MRI.

Measurement of cardiac function is often performed in mice after, for example, a myocardial infarction. Cardiac MRI is often used because it is noninvasive and provides high temporal and spatial resolution for the left and right ventricle. In animal cardiac MRI, the quality of the required electrocardiogram signal is variable and sometimes deteriorates over time, especially with infarcted hearts or cardiac hypertrophy. Therefore, we compared the self-gated IntraGateFLASH method with a prospectively triggered FLASH (fast low-angle shot) method in mice with myocardial infarcts (n = 16) and in control mice (n = 21). Mice with a myocardial infarct and control mice were imaged in a vertical 9.4-T MR system. Images of contiguous 1-mm slices were acquired from apex to base with prospective and self-gated methods. Data were processed to calculate cardiac function parameters for the left and right ventricle. The signal-to-noise and contrast-to-noise ratios were calculated in mid-ventricular slices. The signal-to-noise and contrast-to-noise ratios of the self-gated data were higher than those of the prospectively gated data. Differences between the two gating methods in the cardiac function parameters for both left and right ventricle (e.g. end-diastolic volumes) did not exceed the inter-observer variability in control or myocardial infarcted mice. Both methods gave comparable results with regard to the cardiac function parameters in both healthy control mice and mice with myocardial infarcts. Moreover, the self-gated method provided better signal-to-noise and contrast-to-noise ratios when the acquisition time was equal. In conclusion, the self-gated method is suitable for routine use in cardiac MRI in mice with myocardial infarcts as well as in control mice, and obviates the need for electrocardiogram triggering and respiratory gating. In both gating methods, more than 10 frames per cardiac cycle are recommended.

Targeted deletion of nuclear factor kappaB p50 enhances cardiac remodeling and dysfunction following myocardial infarction.

Myocardial infarction is commonly complicated by left ventricular remodeling, a process that leads to cardiac dilatation, congestive heart failure and death. The innate immune system plays a pivotal role in the remodeling process via nuclear factor (NF)-kappaB activation. The NF-kappaB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. The function of NF-kappaB p50, however, is controversial in this process. To clarify the role of NF-kappaB p50 in postinfarct left ventricular remodeling, myocardial infarction was induced in wild-type 129Bl6 mice and NF-kappaB p50-deficient mice. Without affecting infarct size, deletion of NF-kappaB p50 markedly increased the extent of expansive remodeling (end-diastolic volume: 176+/-13 microL versus 107+/-11 microL; P=0.003) and aggravated systolic dysfunction (left ventricular ejection fraction: 16.1+/-1.5% versus 24.7+/-3.7%; P=0.029) in a 28-day time period. Interstitial fibrosis and hypertrophy in the noninfarcted myocardium was increased in NF-kappaB p50 knockout mice. In the infarct area, a lower collagen density was observed, which was accompanied by an increased number of macrophages, higher gelatinase activity and increased inflammatory cytokine expression. In conclusion, targeted deletion of NF-kappaB p50 results in enhanced cardiac remodeling and functional deterioration following myocardial infarction by increasing matrix remodeling and inflammation.

Modulation of TGF-ß/BMP-6 expression and increased levels of circulating smooth muscle progenitor cells in a type I diabetes mouse model.

BACKGROUND: Diabetic patients experience exaggerated intimal hyperplasia after endovascular procedures. Recently it has been shown that circulating smooth muscle progenitor cells (SPC) contribute to intimal hyperplasia. We hypothesized that SPC differentiation would be increased in diabetes and focused on modulation of TGF-ß/BMP-6 signaling as potential underlying mechanism. METHODS: We isolated SPC from C57Bl/6 mice with streptozotocin-induced diabetes and controls. SPC differentiation was evaluated by immunofluorescent staining for αSMA and collagen Type I. SPC mRNA expression of TGF-ß and BMP-6 was quantified using real-time PCR. Intima formation was assessed in cuffed femoral arteries. Homing of bone marrow derived cells to cuffed arterial segments was evaluated in animals transplanted with bone marrow from GFP-transgenic mice. RESULTS: We observed that SPC differentiation was accelerated and numeric outgrowth increased in diabetic animals (24.6 ± 8.8 vs 8.3 ± 1.9 per HPF after 10 days, p < 0.05). Quantitative real-time PCR showed increased expression of TGF-ß and decreased expression of the BMP-6 in diabetic SPC. SPC were MAC-3 positive, indicative of monocytic lineage. Intima formation in cuffed arterial segments was increased in diabetic mice (intima/media ratio 0.68 ± 0.15 vs 0.29 ± 0.06, p < 0.05). In GFP-chimeric mice, bone marrow derived cells were observed in the neointima (4.4 ± 3.3 cells per section) and particularly in the adventitia (43.6 ± 9.3 cells per section). GFP-positive cells were in part MAC-3 positive, but rarely expressed α-SMA. CONCLUSIONS: In conclusion, in a diabetic mouse model, SPC levels are increased and SPC TGF-ß/BMP-6 expression is modulated. Altered TGF-ß/BMP-6 expression is known to regulate smooth muscle cell differentiation and may facilitate SPC differentiation. This may contribute to exaggerated intimal hyperplasia in diabetes as bone marrow derived cells home to sites of neointima formation.

Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury.

Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI.

DNA replication stress underlies renal phenotypes in CEP290-associated Joubert syndrome.

Juvenile ciliopathy syndromes that are associated with renal cysts and premature renal failure are commonly the result of mutations in the gene encoding centrosomal protein CEP290. In addition to centrosomes and the transition zone at the base of the primary cilium, CEP290 also localizes to the nucleus; however, the nuclear function of CEP290 is unknown. Here, we demonstrate that reduction of cellular CEP290 in primary human and mouse kidney cells as well as in zebrafish embryos leads to enhanced DNA damage signaling and accumulation of DNA breaks ex vivo and in vivo. Compared with those from WT mice, primary kidney cells from Cep290-deficient mice exhibited supernumerary centrioles, decreased replication fork velocity, fork asymmetry, and increased levels of cyclin-dependent kinases (CDKs). Treatment of Cep290-deficient cells with CDK inhibitors rescued DNA damage and centriole number. Moreover, the loss of primary cilia that results from CEP290 dysfunction was rescued in 3D cell culture spheroids of primary murine kidney cells after exposure to CDK inhibitors. Together, our results provide a link between CEP290 and DNA replication stress and suggest CDK inhibition as a potential treatment strategy for a wide range of ciliopathy syndromes.

Development of a cryopreservation protocol for type A spermatogonia.

The aim of this study was to develop a cryopreservation protocol for type A spermatogonia. Testes from 5- to 7-month-old calves were collected, and type A spermatogonia were isolated using two-step enzymatic digestion and Percoll separation. Cells were resuspended in minimum essential medium (MEM) supplemented with 1% bovine serum albumin (BSA) in a final concentration of 6 x 10(6) per mL, and the effects of different cryoprotectants and freezing protocols were tested. Cells frozen/thawed in medium containing 10% fetal calf serum (FCS) and 1.4 M glycerol or dimethyl sulfoxide (DMSO) had a significantly (P <.05) higher percentage of living cells compared to medium with only FCS, whereas DMSO gave a significantly better cell survival rate than glycerol did. An increase in the concentration of FCS in the DMSO-based medium to 20% had no effect on survival after freezing and thawing. Furthermore, inclusion of 0.07, 0.14, or 0.21 M sucrose in DMSO-based medium resulted in a significant improvement of cell survival, cell proliferation in culture, and colonization efficiency in recipient testes. A controlled slow-freezing rate (1 degrees C/min) resulted in significantly (P <.05) more viable cells than fast (5 degrees C/min) freezing. However, noncontrolled-rate freezing, with a comparably low cooling rate, gave even better results than the controlled-rate slow freezing. Cryopreservation in MEM-based medium containing 10% FCS, 10% DMSO, and 0.07 M sucrose using a non-controlled-rate freezing protocol appeared to be a simple and effective way to preserve type A spermatogonia, with a high yield of almost 70% living cells after thawing. Frozen/thawed spermatogonia survived in culture and retained the ability to proliferate as determined by colorimetric and bromodeoxyuridine incorporation assays. To test whether the stem cells among the A spermatogonia retained their ability to colonize the testis of a recipient mouse, bovine spermatogonia were transplanted. This resulted in colonization 2-3 months after transplantation. In conclusion, for the first time, a method specific for cryopreservation of type A spermatogonia, including spermatogonial stem cells was developed, which allows long-term preservation of these cells without apparent harmful effects to their function.

Increasing short-term cardiomyocyte progenitor cell (CMPC) survival by necrostatin-1 did not further preserve cardiac function.

AIMS: One of the main limitations for an effective cell therapy for the heart is the poor cell engraftment after implantation, which is partly due to a large percentage of cell death in the hostile myocardium. In the present study, we investigated the utilization of necrostatin-1 (Nec-1) as a possible attenuator of cell death in cardiomyocyte progenitor cells (CMPCs). METHODS AND RESULTS: In a mouse model of myocardial infarction, survival of CMPCs 3 days after intra-myocardial injection was 39 ± 9% higher in cells pretreated with the Nec-1 compound. However, the increase in cell number was not sustained over 28 days, and did not translate into improved cardiac function (ejection fraction %, 20.6 ± 2.1 vs. 21.4 ± 2.5 for vehicle and Nec-1-treated CMPC, respectively). Nonetheless, Nec-1 rescued CMPCs remained functionally competent. CONCLUSION: A pharmacological pretreatment approach to solely enhance cell survival on the short term does not seem to be effective strategy to improve cardiac cell therapy with CMPCs.

ACE Inhibition in Anti-Thy1 Glomerulonephritis Limits Proteinuria but Does Not Improve Renal Function and Structural Remodeling.

BACKGROUND/AIMS: ACE inhibitor (ACE-I) treatment effectively inhibits proteinuria and ameliorates the course of various renal diseases. In experimental glomerulonephritis, however, angiotensin II (AngII) infusion has also been shown to be renoprotective. We evaluated the long-term (28 days) course of anti-Thy1 glomerulonephritis in animals with suppressed AngII formation by ACE-I treatment. METHODS: Brown Norway rats received perindopril (2.8 mg/kg/day, n = 12), dihydropyridine calcium-antagonist amlodipine (Ca-A; 13 mg/kg/day, n = 6) or were left untreated (n = 14). All animals were monitored for blood pressure, proteinuria, and creatinine clearance after anti-Thy1 injection. Renal histology was assessed at day 7 and 28. RESULTS: Systolic blood pressure was equally reduced by ACE-I and Ca-A treatment. AngII suppression prevented development of proteinuria, but did not protect against glomerular microaneurysm formation or reduction in creatinine clearance. After resolution of the microaneurysms, animals with suppressed AngII production showed a modest increase in glomerulosclerosis and vasculopathic thickening of intrarenal vessels. CONCLUSIONS: In anti-Thy1 glomerulonephritis, suppression of AngII formation does not protect against the induction of glomerular damage and is associated with mild aggravation of adverse renal fibrotic remodeling. Proteinuria, however, is effectively prevented by ACE-I treatment. Ca-A treatment did not affect the course of glomerulonephritis, indicating that ACE-I effects are blood pressure independent.

Human cardiomyocyte progenitor cell transplantation preserves long-term function of the infarcted mouse myocardium.

AIMS: Recent clinical studies revealed that positive results of cell transplantation on cardiac function are limited to the short- and mid-term restoration phase following myocardial infarction (MI), emphasizing the need for long-term follow-up. These transient effects may depend on the transplanted cell-type or its differentiation state. We have identified a population of cardiomyocyte progenitor cells (CMPCs) capable of differentiating efficiently into beating cardiomyocytes, endothelial cells, and smooth muscle cells in vitro. We investigated whether CMPCs or pre-differentiated CMPC-derived cardiomyocytes (CMPC-CM) are able to restore the injured myocardium after MI in mice. METHODS AND RESULTS: MI was induced in immunodeficient mice and was followed by intra-myocardial injection of CMPCs, CMPC-CM, or vehicle. Cardiac function was measured longitudinally up to 3 months post-MI using 9.4 Tesla magnetic resonance imaging. The fate of the human cells was determined by immunohistochemistry. Transplantation of CMPCs or CMPC-CM resulted in a higher ejection fraction and reduced the extent of left ventricular remodelling up to 3 months after MI when compared with vehicle-injected animals. CMPCs and CMPC-CM generated new cardiac tissue consisting of human cardiomyocytes and blood vessels. Fusion of human nuclei with murine nuclei was not observed. CONCLUSION: CMPCs differentiated into the same cell types in situ as can be obtained in vitro. This excludes the need for in vitro pre-differentiation, making CMPCs a promising source for (autologous) cell-based therapy.

Improvement of mouse cardiac function by hESC-derived cardiomyocytes correlates with vascularity but not graft size.

Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) has been shown to improve the function of the rodent heart 1 month after myocardial infarction (MI). However, the mechanistic basis and optimal delivery strategies are unclear. We investigated the influence of the number of injected cells, resulting graft size, and possible paracrine mechanisms in this process. MI was induced in NOD-SCID mice (n=84) followed by injection of enriched hESC-CM at different dosages, hESC-non-CM derivatives, culture medium, or no injection. Cardiac function was monitored for 12 weeks with 9.4 T MRI (n=70). Grafts were identified by epifluorescence of a transgenic GFP marker and characterized by immunofluorescence. Vascularity and paracrine effects were investigated immunohistochemically. Transplantation of differentiated hESCs improved short, mid-, and long-term cardiac performance and survival, although only cardiomyocytes formed grafts. A mid-term (4 weeks) cardiomyocyte-specific enhancement was associated with elevated vascular density around the graft and attenuated compensatory remodeling. However, increasing the number of hESC-CM for injection did not enhance heart function further. Moreover, we observed that small graft size was associated with a better functional outcome. HESC-CM increased myocardial vascularization and enhanced heart function in mice after MI, but larger graft size was associated with reduced functional improvement. Future studies should focus on advanced delivery strategies and mechanisms of action rather than increasing graft size.

Amelioration of anti-Thy1-glomerulonephritis by PPAR-gamma agonism without increase of endothelial progenitor cell homing.

Impaired glomerular endothelial integrity is pivotal in various renal diseases and depends on both the degree of glomerular endothelial injury and the effectiveness of glomerular endothelial repair. Glomerular endothelial repair is, in part, mediated by bone marrow-derived endothelial progenitor cells. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists have therapeutic actions independent of their insulin-sensitizing effects, including enhancement of endothelial progenitor cell function and differentiation. We evaluated the effect of PPAR-gamma agonist rosiglitazone (4 mg.kg(-1).day(-1)) on the course of anti-Thy1-glomerulonephritis in rats. Rosiglitazone limited the development of proteinuria and prevented plasma urea elevation (8.1 +/- 0.4 vs. 12.5 +/- 1.1 mmol/l, P = 0.002). Histologically, inflammatory cell influx was not affected, but rosiglitazone-treated rats did show fewer microaneurysmatic glomeruli on day 7 (26 +/- 3 vs. 41 +/- 5%, P = 0.01) and reduced activation of matrix production with reduced renal cortical transforming growth factor-beta, plasminogen activator inhibitor type 1, and fibronectin-1 mRNA expression. However, bone marrow-derived endothelial cell glomerular incorporation was not enhanced (3.1 +/- 0.4 vs. 3.6 +/- 0.3 cells/glomerular cross section; P = 0.31). Rosiglitazone treatment in nonnephritic rats did not influence proteinuria, urea, or renal histology. In conclusion, treatment with PPAR-gamma agonist rosiglitazone ameliorates the course of experimental glomerulonephritis in a nondiabetic model, but not through enhancing incorporation of bone marrow-derived endothelial cells in the glomerulus.

Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction.

Regeneration of the myocardium by transplantation of cardiomyocytes is an emerging therapeutic strategy. Human embryonic stem cells (HESC) form cardiomyocytes readily but until recently at low efficiency, so that preclinical studies on transplantation in animals are only just beginning. Here, we show the results of the first long-term (12 weeks) analysis of the fate of HESC-derived cardiomyocytes transplanted intramyocardially into healthy, immunocompromised (NOD-SCID) mice and in NOD-SCID mice that had undergone myocardial infarction (MI). Transplantation of mixed populations of differentiated HESC containing 20-25% cardiomyocytes in control mice resulted in rapid formation of grafts in which the cardiomyocytes became organized and matured over time and the noncardiomyocyte population was lost. Grafts also formed in mice that had undergone MI. Four weeks after transplantation and MI, this resulted in significant improvement in cardiac function measured by magnetic resonance imaging. However, at 12 weeks, this was not sustained despite graft survival. This suggested that graft size was still limiting despite maturation and organization of the transplanted cells. More generally, the results argued for requiring a minimum of 3 months follow-up in studies claiming to observe improved cardiac function, independent of whether HESC or other (adult) cell types are used for transplantation.