Topical application of cannabinoid-ligands ameliorates experimental dry-eye disease.

PURPOSE: Dry eye disease (DED) is a multifactorial disease, with limitations regarding efficacy and tolerability of applied substances. Among several candidates, the endocannabinoid system with its receptors (CB1R and CB2R) were reported to modulate inflammation, wound healing and pain, which are also core DED pathomechanisms. This study is to investigate the therapeutic responses of Δ-9 tetrahydrocannabinol (a non-selective agonist) and two selective antagonists, SR141716A (CB1R antagonist) and SR144528 (CB2R antagonist), as a topical application using a DED mouse model. METHOD: Experimental DED was induced in naïve C57BL/6 mice. Expression of CBR at the ocular surface of naïve and DED mice was determined by qPCR and in-situ hybridization. Either THC or CBR antagonists were compounded in an aqueous solution and dosed during the induction of DED. Tear production, cornea sensitivity, and cornea fluorescence staining were tested. At the end of each experiment, corneas were stained with ß3-tubulin for analysis of corneal nerve morphology. Conjunctiva was analyzed for CD4+ and CD8+ infiltration. RESULTS: CB1R and CB2R are present at the ocular surface, and desiccating stress increased CBR expressions (p < 0.05). After 10 days of DED induction, treated groups demonstrated a reduced CBR expression in the cornea, which was concurrent with improvements in the DED phenotype including fluorescence staining & inflammation. Applying THC protected corneal nerve morphology, thus maintained corneal sensitivity and reduced CD4+ T-cell infiltration. The CB1R antagonist maintained cornea sensitivity without changing nerve morphology. CONCLUSIONS: Endocannabinoid receptor modulation presents a potential multi-functional therapeutic approach for DED.

Protective Effects of Therapeutic Neutrophil Depletion and Myeloperoxidase Inhibition on Left Ventricular Function and Remodeling in Myocardial Infarction.

Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. Improved survival has led to an increasing incidence of ischemic cardiomyopathy, making it a major reason for hospitalization in the western world. The inflammatory response in the ischemic myocardium determines the extent of structural remodeling and functional deterioration, with neutrophils (PMN) being a key modulator of the propagation and resolution of inflammation. The heme enzyme myeloperoxidase (MPO) is abundantly expressed in PMN and is an important mediator of their inflammatory capacities. Here, we examine the effects of PMN reduction, MPO deficiency and MPO inhibition in two murine models of MI. Reduction in PMN count resulted in less scar formation and improved cardiac function. Similar results were obtained in genetically MPO deficient mice, suggesting that MPO is a critical factor in PMN-mediated cardiac remodeling. To test our findings in a therapeutic approach, we orally administered the MPO inhibitor AZM198 in the context of MI and could demonstrate improved cardiac function and reduced structural remodeling. Therefore, MPO appears to be a favorable pharmacological target for the prevention of long-term morbidity after MI.

Stamp2 Protects From Maladaptive Structural Remodeling and Systolic Dysfunction in Post-Ischemic Hearts by Attenuating Neutrophil Activation.

The six-transmembrane protein of prostate 2 (Stamp2) acts as an anti-inflammatory protein in macrophages by protecting from overt inflammatory signaling and Stamp2 deficiency accelerates atherosclerosis in mice. Herein, we describe an unexpected role of Stamp2 in polymorphonuclear neutrophils (PMN) and characterize Stamp2's protective effects in myocardial ischemic injury. In a murine model of ischemia and reperfusion (I/R), echocardiography and histological analyses revealed a pronounced impairment of cardiac function in hearts of Stamp2-deficient- (Stamp2-/- ) mice as compared to wild-type (WT) animals. This difference was driven by aggravated cardiac fibrosis, as augmented fibroblast-to-myofibroblast transdifferentiation was observed which was mediated by activation of the redox-sensitive p38 mitogen-activated protein kinase (p38 MAPK). Furthermore, we observed increased production of reactive oxygen species (ROS) in Stamp2-/- hearts after I/R, which is the likely cause for p38 MAPK activation. Although myocardial macrophage numbers were not affected by Stamp2 deficiency after I/R, augmented myocardial infiltration by polymorphonuclear neutrophils (PMN) was observed, which coincided with enhanced myeloperoxidase (MPO) plasma levels. Primary PMN isolated from Stamp2-/- animals exhibited a proinflammatory phenotype characterized by enhanced nuclear factor (NF)-κB activity and MPO secretion. To prove the critical role of PMN for the observed phenotype after I/R, antibody-mediated PMN depletion was performed in Stamp2-/- mice which reduced deterioration of LV function and adverse structural remodeling to WT levels. These data indicate a novel role of Stamp2 as an anti-inflammatory regulator of PMN and fibroblast-to-myofibroblast transdifferentiation in myocardial I/R injury.

Ocular Graft-versus-Host Disease in a Chemotherapy-Based Minor-Mismatch Mouse Model Features Corneal (Lymph-) Angiogenesis.

Ocular graft-versus-host disease (oGVHD) is a fast progressing, autoimmunological disease following hematopoietic stem cell transplantation, leading to severe inflammation of the eye and destruction of the lacrimal functional unit with consecutive sight-threatening consequences. The therapeutic "window of opportunity" is narrow, and current treatment options are limited and often insufficient. To achieve new insights into the pathogenesis and to develop new therapeutic approaches, clinically relevant models of oGVHD are desirable. In this study, the ocular phenotype was described in a murine, chemotherapy-based, minor-mismatch GVHD model mimicking early-onset chronic oGVHD, with corneal epitheliopathy, inflammation of the lacrimal glands, and blepharitis. Additionally, corneal lymphangiogenesis was observed as part of oGVHD pathogenesis for the first time, thus opening up the investigation of lymphangiogenesis as a potential therapeutic and diagnostic tool.

Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages.

BACKGROUND: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. METHODS: To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6-7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. RESULTS: The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. CONCLUSION: The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

In Vitro Grown Micro-Tissues for Cardiac Cell Replacement Therapy in Vivo.

BACKGROUND/AIMS: Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice. METHODS: Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections. RESULTS: We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging. CONCLUSION: This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.

Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling.

RATIONALE: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. OBJECTIVE: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. METHODS AND RESULTS: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin-deficient (CD11b-/-) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo-/-) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo-/- mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell-derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo-/- mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter-defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. CONCLUSIONS: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.

Intramyocardially Transplanted Neonatal Cardiomyocytes (NCMs) Show Structural and Electrophysiological Maturation and Integration and Dose-Dependently Stabilize Function of Infarcted Rat Hearts.

Cardiac cell replacement therapy is a promising therapy to improve cardiac function in heart failure. Persistence, structural and functional maturation, and integration of transplanted cardiomyocytes into recipients' hearts are crucial for a safe and efficient replacement of lost cells. We studied histology, electrophysiology, and quantity of intramyocardially transplanted rat neonatal cardiomyocytes (NCMs) and performed a detailed functional study with repeated invasive (pressure-volume catheter) and noninvasive (echocardiography) analyses of infarcted female rat hearts including pharmacological stress before and 3 weeks after intramyocardial injection of 5 × 106 (low NCM) or 25 × 106 (high NCM) syngeneic male NCMs or medium as placebo (Ctrl). Quantitative real-time polymerase chain reaction (PCR) for Y-chromosome confirmed a fivefold higher persisting male cell number in high NCM versus low NCM after 3 weeks. Sharp electrode measurements within viable slices of recipient hearts demonstrated that transplanted NCMs integrate into host myocardium and mature to an almost adult phenotype, which might be facilitated through gap junctions between host myocardium and transplanted NCMs as indicated by connexin43 in histology. Ejection fraction of recipient hearts was severely impaired after ligation of left anterior descending (LAD; pressure-volume catheter: 39.2 ± 3.6%, echocardiography: 39.9 ± 1.4%). Repeated analyses revealed a significant further decline within 3 weeks in Ctrl and a dose-dependent stabilization in cell-treated groups. Consistently, stabilized cardiac function/morphology in cell-treated groups was seen in stroke volume, cardiac output, ventricle length, and wall thickness. Our findings confirm that cardiac cell replacement is a promising therapy for ischemic heart disease since immature cardiomyocytes persist, integrate, and mature after intramyocardial transplantation, and they dose-dependently stabilize cardiac function after myocardial infarction.

Lack of Gαi2 leads to dilative cardiomyopathy and increased mortality in ß1-adrenoceptor overexpressing mice.

AIMS: Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac ß1-adrenoceptor overexpression. METHODS AND RESULTS: ß1-transgenic mice lacking Gαi2 (ß1-tg/Gαi2 (-/-)) were compared with wild-type mice and littermates either overexpressing cardiac ß1-adrenoceptors (ß1-tg) or lacking Gαi2 (Gαi2 (-/-)). At 300 days, mortality of mice only lacking Gαi2 was already higher compared with wild-type or ß1-tg, but similar to ß1-tg/Gαi2 (-/-), mice. Beyond 300 days, mortality of ß1-tg/Gαi2 (-/-) mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, ß1-tg, and Gαi2 (-/-) mice, but significant impairment for ß1-tg/Gαi2 (-/-) mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gαi3 was significantly up-regulated in Gαi2 knockout mice (protein compared with wild type: 340 ± 90% in Gαi2 (-/-) and 394 ± 80% in ß1-tg/Gαi2 (-/-), respectively). CONCLUSIONS: Gαi2 deficiency combined with cardiac ß1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, ß1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased ß1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism.

Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C-terminal phosphorylation as a determinant of slit diaphragm complex architecture.

Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross-species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph-1 (Kirrel). Moreover, cross-species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology. All MS data have been deposited in the ProteomeXchange with identifier PXD001005 (http://proteomecentral.proteomexchange.org/dataset/PXD001005).

Electrophysiological and morphological maturation of murine fetal cardiomyocytes during electrical stimulation in vitro.

The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and cell morphology of fetal cardiomyocytes (FCMs) in culture. Fetal cardiomyocytes at day 14.5 post coitum were harvested from murine hearts and electrically stimulated for 6 days in culture using a custom-made stimulation chamber. Subsequently, action potentials of FCM were recorded with glass microelectrodes. Immunostainings of α-Actinin, connexin 43, and vinculin were performed. Expression of ion channel subunits Kcnd2, Slc8a1, Cacna1, Kcnh2, and Kcnb1 was analyzed by quantitative reverse-transcriptase polymerase chain reaction. Action potential duration to 50% and 90% repolarization (APD50 and APD90) of electrically stimulated FCMs were significantly decreased when compared to nonstimulated control FCM. Alignment of cells was significantly higher in stimulated FCM when compared to control FCM. The expression of connexin 43 was significantly increased in stimulated FCM when compared to control FCM. The ratio between cell length and cell width of the stimulated FCM was significantly higher than in control FCM. Kcnh2 and Kcnd2 were upregulated in stimulated FCM when compared to control FCM. Expression of Slc8a1, Cacna1c, and Kcnb1 was not different in stimulated and control FCMs. The decrease in APD50 observed after electrical stimulation of FCM in vitro corresponds to the electrophysiological maturation of FCM in vivo. Expression levels of ion channels suggest that some important but not all aspects of the complex process of electrophysiological maturation are promoted by electrical stimulation. Parallel alignment, increased connexin 43 expression, and elongation of FCM are signs of a morphological maturation induced by electrical stimulation.

Electrophysiological integration and action potential properties of transplanted cardiomyocytes derived from induced pluripotent stem cells.

AIMS: Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) are regarded as promising cell type for cardiac cell replacement therapy. We investigated long-term electrophysiological integration and maturation of transplanted iPSCM, which are essential for therapeutic benefit. METHODS AND RESULTS: Murine iPSCM expressing enhanced green fluorescent protein and a puromycin resistance under control of the α-myosin heavy chain promoter were purified by antibiotic selection and injected into adult mouse hearts. After 6-12 days, 3-6 weeks, or 6-8 months, viable slices of recipient hearts were prepared. Slices were focally stimulated by a unipolar electrode placed in host tissue, and intracellular action potentials (APs) were recorded with glass microelectrodes in transplanted cells and neighbouring host tissue within the slices. Persistence and electrical integration of transplanted iPSCM into recipient hearts could be demonstrated at all time points. Quality of coupling improved, as indicated by a maximal stimulation frequency without conduction blocks of 5.77 ± 0.54 Hz at 6-12 days, 8.98 ± 0.38 Hz at 3-6 weeks and 10.82 ± 1.07 Hz at 6-8 months after transplantation. AP properties of iPSCM became more mature from 6-12 days to 6-8 months after transplantation, but still differed significantly from those of host APs. CONCLUSION: Transplanted iPSCM can persist in the long term and integrate electrically into host tissue, supporting their potential for cell replacement therapy. Quality of electrical integration improves between 6-12 days and 6-8 months after transplantation, and there are signs of an electrophysiological maturation. However, even after 6-8 months, AP properties of transplanted iPSCM differ from those of recipient cardiomyocytes.