Decreased or impaired proliferation capability of dermal fibroblasts interferes with successful wound healing. Several growth factors tested failed to fully restore the growth of fibroblasts, possibly due to their rapid degradation by proteases. It is therefore critical to find new agents which have stimulatory effects on fibroblasts while being highly resistant to degradation. In such a scenario, the activities of two agonistic analogs of growth hormone releasing hormone (GHRH), MR-409 and MR-502, were evaluated for their impact on proliferation and survival of primary human dermal fibroblasts. In vitro, both analogs significantly stimulated cell growth by more than 50%. Under serum-depletion induced stress, fibroblasts treated with MR-409 or MR-502 demonstrated better survival rates than control. These effects can be inhibited by either PD98059 or wortmannin. Signaling through MEK/ERK1/2 and PI3K/AKT in an IGF-1 receptor-independent manner is required. In vivo, MR-409 promoted wound closure. Animals treated topically with MR-409 healed earlier than controls in a dose-dependent manner. Histologic examination revealed better wound contraction and less fibrosis in treated groups. In conclusion, MR-409 is a potent mitogenic and anti-apoptotic factor for primary human dermal fibroblasts. Its beneficial effects on wound healing make it a promising agent for future development.

Uveal melanoma (UM) is the most common primary intraocular malignancy with very poor prognosis. Conventional chemotherapy only rarely prolongs the survival, therefore patients require novel treatment modalities. The discovery of specific receptors for hypothalamic hormones on cancer cells has led to the development of radiolabeled and cytotoxic hormone analogs.

Management of castration-resistant prostate cancer (CRPC) is challenging due to lack of efficacious therapy. Luteinizing hormone-releasing hormone analogs appear to act directly on cells based on the LHRH receptors on human prostate adenocarcinoma cells. We explored anticancer activity of a cytotoxic analog of LHRH, AEZS-108 consisting of LHRH agonist linked to doxorubicin. Nude mice bearing DU-145 tumors were used to compare antitumor effects of AEZS-108 with its individual constituents or their unconjugated combination. The tumor growth inhibition of conjugate was greatest among treatment groups (90.5% inhibition vs. 41% by [D-Lys(6)]LHRH+DOX). The presence of LHRH receptors on DU-145 cells was confirmed by immunocytochemistry. In vitro, AEZS-108 significantly inhibited cell proliferation (61.2% inhibition) and elevated apoptosis rates (by 46%). By the detection of the inherent doxorubicin fluorescence, unconjugated doxorubicin was seen in the nucleus; the conjugate was perinuclear and at cell membrane. Autophagy, visualized by GFP-tagged p62 reporter, was increased by AEZS-108 (7.9-fold vs. 5.3-fold by DOX+[D-Lys(6)]LHRH. AEZS-108 more effectively increased reactive oxygen species (ROS, 2-fold vs. 1.4-fold by DOX+[D-Lys(6)]LHRH) and levels of the apoptotic regulator p21 in vivo and in vitro. We demonstrate robust inhibitory effects of the targeted cytotoxic LHRH analog AEZS-108 on LHRHR positive castration-resistant prostate cancer cells.

The cross-talk between beta cells and endothelium plays a key role in islet physiopathology and in the revascularization process after islet transplantation. However, the molecular mechanisms involved in this cross-talk are not fully elucidated. Extracellular vesicles (EVs) are secreted membrane nanoparticles involved in inter-cellular communication through the transfer of proteins and nucleic acids. The aims of this study were: 1) isolation and characterization of EVs from human islets; 2) evaluation of the pro-angiogenic effect of islet-derived EVs on human islet endothelial cells (IECs). EVs were isolated by ultracentrifugation from conditioned medium of human islets and characterized by nanotrack analysis (Nanosight), FACS, western blot, bioanalyzer, mRNA/microRNA RT-PCR array. On IECs, we evaluated EV-induced insulin mRNA transfer, proliferation, resistance to apoptosis, in vitro angiogenesis, migration, gene and protein profiling. EVs sized 236±54 nm, expressed different surface molecules and islet-specific proteins (insulin, C-peptide, GLP1R) and carried several mRNAs (VEGFa, eNOS) and microRNAs (miR-27b, miR-126, miR-130 and miR-296) involved in beta cell function, insulin secretion and angiogenesis. Purified EVs were internalized into IECs inducing insulin mRNA expression, protection from apoptosis and enhancement of angiogenesis. Human islets release biologically active EVs able to shuttle specific mRNAs and microRNAs (miRNAs) into target endothelial cells. These results suggest a putative role for islet-derived EVs in beta cell-endothelium cross-talk and in the neoangiogenesis process which is critical for engraftment of transplanted islets.

Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia.

Extrapituitary roles for hypothalamic neurohormones have recently become apparent and clinically relevant, based on the use of synthetic peptide analogs for the treatment of multiple conditions including cancers, pulmonary edema and myocardial infarction. In the eye, it has been suggested that some of these hormones and their receptors may be present in the ciliary body, iris, trabecular meshwork and retina, but their physiological role has yet to be elucidated. Our study intends to comprehensively demonstrate the expression of some hypothalamic neuroendocrine hormones and their receptors within different retinal and extraretinal structures of the human eye. Immunofluorescence, Western blot analysis, and RT-PCR were used to evaluate the qualitative and quantitative expression of Luteinizing Hormone Releasing Hormone (LHRH), Growth Hormone Releasing Hormone (GHRH), Thyrotropin Releasing Hormone (TRH), Gastrin Releasing Peptide (GRP) and Somatostatin as well as their respective receptors (LHRH-R, GHRH-R, TRH-R, GRP-R, SST-R1) in cadaveric human eye tissue and in paraffinized human eye tissue sections. The hypothalamic hormones LHRH, GHRH, TRH, GRP and Somatostatin and their respective receptors (LHRH-R, GHRH-R, TRH-R, GRPR/BB2 and SST-R1), were expressed in the conjunctiva, cornea, trabecular meshwork, ciliary body, lens, retina, and optic nerve.

It has been shown that growth hormone-releasing hormone (GHRH) reduces cardiomyocyte (CM) apoptosis, prevents ischemia/reperfusion injury, and improves cardiac function in ischemic rat hearts. However, it is still not known whether GHRH would be beneficial for life-threatening pathological conditions, like cardiac hypertrophy and heart failure (HF). Thus, we tested the myocardial therapeutic potential of GHRH stimulation in vitro and in vivo, using GHRH or its agonistic analog MR-409. We show that in vitro, GHRH(1-44)NH2 attenuates phenylephrine-induced hypertrophy in H9c2 cardiac cells, adult rat ventricular myocytes, and human induced pluripotent stem cell-derived CMs, decreasing expression of hypertrophic genes and regulating hypertrophic pathways. Underlying mechanisms included blockade of Gq signaling and its downstream components phospholipase Cβ, protein kinase Cε, calcineurin, and phospholamban. The receptor-dependent effects of GHRH also involved activation of Gαs and cAMP/PKA, and inhibition of increase in exchange protein directly activated by cAMP1 (Epac1). In vivo, MR-409 mitigated cardiac hypertrophy in mice subjected to transverse aortic constriction and improved cardiac function. Moreover, CMs isolated from transverse aortic constriction mice treated with MR-409 showed improved contractility and reversal of sarcolemmal structure. Overall, these results identify GHRH as an antihypertrophic regulator, underlying its therapeutic potential for HF, and suggest possible beneficial use of its analogs for treatment of pathological cardiac hypertrophy.

Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs).

A pro-apoptotic effect of circulating mediators on renal tubular epithelial cells has been involved in the pathogenesis of sepsis-associated acute kidney injury (AKI). Adsorption techniques have been showed to efficiently remove inflammatory cytokines from plasma. The aim of this study was to evaluate the efficiency of the hydrophobic resin Amberchrom CG161 M to adsorb from septic plasma soluble mediators involved in tubular injury.

Antagonists of growth hormone-releasing hormone (GHRH) are being developed for the treatment of various human cancers.

Therapeutic strategies should be designed to transform aggressive prostate cancer phenotypes to a chronic situation. To evaluate the effects of the new growth hormone-releasing hormone receptor (GHRH-R) antagonists: MIA-602, MIA-606, and MIA-690 on processes associated with cancer progression as cell proliferation, adhesion, migration, and angiogenesis.

Several studies have suggested that extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) may mediate MSC paracrine action on kidney regeneration. This activity has been, at least in part, ascribed to the transfer of proteins/transcription factors and different RNA species. Information on the RNA/protein content of different MSC EV subpopulations and the correlation with their biological activity is currently incomplete. The aim of this study was to evaluate the molecular composition and the functional properties on renal target cells of MSC EV sub-populations separated by gradient floatation. The results demonstrated heterogeneity in quantity and composition of MSC EVs. Two peaks of diameter were observed (90-110 and 170-190 nm). The distribution of exosomal markers and miRNAs evaluated in the twelve gradient fractions showed an enrichment in fractions with a flotation density of 1.08-1.14 g/mL. Based on this observation, we evaluated the biological activity on renal cell proliferation and apoptosis resistance of low (CF1), medium (CF2) and high (CF3) floatation density fractions. EVs derived from all fractions, were internalized by renal cells, CF1 and CF2 but not CF3 fraction stimulated significant cell proliferation. CF2 also inhibited apoptosis on renal tubular cells submitted to ischemia-reperfusion injury. Comparative miRNomic and proteomic profiles reveal a cluster of miRNAs and proteins common to all three fractions and an enrichment of selected molecules related to renal regeneration in CF2 fraction. In conclusion, the CF2 fraction enriched in exosomal markers was the most active on renal tubular cell proliferation and protection from apoptosis.

Dent disease (DD), an X-linked renal tubulopathy, is mainly caused by loss-of-function mutations in CLCN5 (DD1) and OCRL genes. CLCN5 encodes the ClC-5 antiporter that in proximal tubules (PT) participates in the receptor-mediated endocytosis of low molecular weight proteins. Few studies have analyzed the PT expression of ClC-5 and of megalin and cubilin receptors in DD1 kidney biopsies. About 25% of DD cases lack mutations in either CLCN5 or OCRL genes (DD3), and no other disease genes have been discovered so far. Sanger sequencing was used for CLCN5 gene analysis in 158 unrelated males clinically suspected of having DD. The tubular expression of ClC-5, megalin, and cubilin was assessed by immunolabeling in 10 DD1 kidney biopsies. Whole exome sequencing (WES) was performed in eight DD3 patients. Twenty-three novel CLCN5 mutations were identified. ClC-5, megalin, and cubilin were significantly lower in DD1 than in control biopsies. The tubular expression of ClC-5 when detected was irrespective of the type of mutation. In four DD3 patients, WES revealed 12 potentially pathogenic variants in three novel genes (SLC17A1, SLC9A3, and PDZK1), and in three genes known to be associated with monogenic forms of renal proximal tubulopathies (SLC3A, LRP2, and CUBN). The supposed third Dent disease-causing gene was not discovered.

Cell therapies and derived products have a high potential in aiding tissue and organ repairing and have therefore been considered as potential therapies for treating renal diseases. However, few studies have evaluated the impact of these therapies according to the stage of chronic kidney disease. The aim of this study was to evaluate the renoprotective effect of murine bone marrow mesenchymal stromal cells (BM-MSCs), their extracellular vesicles (EVs) and EVs-depleted conditioned medium (dCM) in an aggressive mouse model of chronic cyclosporine (CsA) nephrotoxicity in a preventive and curative manner.

A pro-thrombotic milieu and a higher risk of thrombotic events were observed in patients with CoronaVirus disease-19 (COVID-19). Accordingly, recent data suggested a beneficial role of low molecular weight heparin (LMWH), but the optimal dosage of this treatment is unknown. We evaluated the association between prophylactic vs. intermediate-to-fully anticoagulant doses of enoxaparin and in-hospital adverse events in patients with COVID-19. We retrospectively included 436 consecutive patients admitted in three Italian hospitals. Outcome according to the use of prophylactic (4000 IU) vs. higher (> 4000 IU) daily dosage of enoxaparin was evaluated. The primary end-point was in-hospital death. Secondary outcome measures were in-hospital cardiovascular death, venous thromboembolism, new-onset acute respiratory distress syndrome (ARDS) and mechanical ventilation. A total of 287 patients (65.8%) were treated with the prophylactic enoxaparin regimen and 149 (34.2%) with a higher dosing regimen. The use of prophylactic enoxaparin dose was associated with a similar incidence of all-cause mortality (25.4% vs. 26.9% with the higher dose; OR at multivariable analysis, including the propensity score: 0.847, 95% CI 0.400-0.1.792; p = 0.664). In the prophylactic dose group, a significantly lower incidence of cardiovascular death (OR 0.165), venous thromboembolism (OR 0.067), new-onset ARDS (OR 0.454) and mechanical intubation (OR 0.150) was observed. In patients hospitalized for COVID-19, the use of a prophylactic dosage of enoxaparin appears to be associated with similar in-hospital overall mortality compared to higher doses. These findings require confirmation in a randomized, controlled study.

Knowledge of the host response to the novel coronavirus SARS-CoV-2 remains limited, hindering the understanding of COVID-19 pathogenesis and the development of therapeutic strategies. During the course of a viral infection, host cells release exosomes and other extracellular vesicles carrying viral and host components that can modulate the immune response. The present study used a shotgun proteomic approach to map the host circulating exosomes' response to SARS-CoV-2 infection. We investigated how SARS-CoV-2 infection modulates exosome content, exosomes' involvement in disease progression, and the potential use of plasma exosomes as biomarkers of disease severity. A proteomic analysis of patient-derived exosomes identified several molecules involved in the immune response, inflammation, and activation of the coagulation and complement pathways, which are the main mechanisms of COVID-19-associated tissue damage and multiple organ dysfunctions. In addition, several potential biomarkers-such as fibrinogen, fibronectin, complement C1r subcomponent and serum amyloid P-component-were shown to have a diagnostic feature presenting an area under the curve (AUC) of almost 1. Proteins correlating with disease severity were also detected. Moreover, for the first time, we identified the presence of SARS-CoV-2 RNA in the exosomal cargo, which suggests that the virus might use the endocytosis route to spread infection. Our findings indicate circulating exosomes' significant contribution to several processes-such as inflammation, coagulation, and immunomodulation-during SARS-CoV-2 infection. The study's data are available via ProteomeXchange with the identifier PXD021144.

The endoplasmic reticulum (ER) stress is one of the mechanisms related to decreased insulin secretion and beta cell death, contributing to the progress of type 2 diabetes mellitus (T2D). Thus, investigating agents that can influence this process would help prevent the development of T2D. Recently, the growth-hormone-releasing hormone (GHRH) action has been demonstrated in INS-1E cells, in which it increases cell proliferation and insulin secretion. As the effects of GHRH and its agonists have not been fully elucidated in the beta cell, we proposed to investigate them by evaluating the role of the GHRH agonist, MR-409, in cells under ER stress. Our results show that the agonist was unable to ameliorate or prevent ER stress. However, cells exposed to the agonist showed less oxidative stress and greater survival even under ER stress. The mechanisms by which GHRH agonist, MR-409, leads to these outcomes require further investigation.

Growth hormone-releasing hormone (GHRH) is a potent stimulator of growth hormone (GH) secretion from the pituitary gland. Although GHRH is essential for the growth of immune cells, the regulatory effects of its antagonist in granulomatous disease remain unknown.

Hematological and oncological disorders represent leading causes of childhood mortality. Neuropeptide somatostatin (SST) has been previously demonstrated in various pediatric tumors, but limited information exists on the expression and characteristics of SST receptors (SSTR) in hematological and oncological disorders of children. We aimed to investigate the expression of mRNA for SSTR subtypes (SSTR-1-5) in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of SSTRs were further studies by ligand competition assay. Our results show that the pediatric tumor samples highly expressed mRNA for the five SSTR subtypes with various patterns. The mRNA for SSTR-2 was detected in all specimens independently of their histological type. A Hodgkin lymphoma sample co-expressed mRNA for all five SSTR subtypes. SSTR-3 and SSTR-5 were detected only in malignant specimens, such as rhabdomyosarcoma, Hodgkin lymphoma, acute lymphoblastic leukemia, and a single nonmalignant condition, hereditary spherocytosis. The incidence of SSTR-1 and SSTR-4 was similar (60%) in the 15 specimens investigated. Radioligand binding studies demonstrated the presence of specific SSTRs and high affinity binding of SST analogs in pediatric solid tumors investigated. The high incidence of SSTRs in hematological and oncological disorders in children supports the merit of further investigation of SSTRs as molecular targets for diagnosis and therapy.

Sepsis-induced acute kidney injury (AKI) is a frequent complication in critically ill patients, refractory to conventional treatments. Aberrant activation of innate immune system may affect organ damage with poor prognosis for septic patients. Here, we investigated the efficacy of polymethyl methacrylate membrane (PMMA)-based continuous hemofiltration (CVVH) in modulating systemic and tissue immune activation in a swine model of LPS-induced AKI. After 3 h from LPS infusion, animals underwent to PMMA-CVVH or polysulfone (PS)-CVVH. Renal deposition of terminal complement mediator C5b-9 and of Pentraxin-3 (PTX3) deposits were evaluated on biopsies whereas systemic Complement activation was assessed by ELISA assay. Gene expression profile was performed from isolated peripheral blood mononuclear cells (PBMC) by microarrays and the results validated by Real-time PCR. Endotoxemic pigs presented oliguric AKI with increased tubulo-interstitial infiltrate, extensive collagen deposition, and glomerular thrombi; local PTX-3 and C5b-9 renal deposits and increased serum activation of classical and alternative Complement pathways were found in endotoxemic animals. PMMA-CVVH treatment significantly reduced tissue and systemic Complement activation limiting renal damage and fibrosis. By microarray analysis, we identified 711 and 913 differentially expressed genes with a fold change >2 and a false discovery rate <0.05 in endotoxemic pigs and PMMA-CVVH treated-animals, respectively. The most modulated genes were Granzyme B, Complement Factor B, Complement Component 4 Binding Protein Alpha, IL-12, and SERPINB-1 that were closely related to sepsis-induced immunological process. Our data suggest that PMMA-based CVVH can efficiently modulate immunological dysfunction in LPS-induced AKI.