Therapeutic application of regeneration-associated cells: a novel source of regenerative medicine.

Chronic diseases with comorbidities or associated risk factors may impair the function of regenerative cells and the regenerative microenvironment. Following this consideration, the vasculogenic conditioning culture (VCC) method was developed to boost the regenerative microenvironment to achieve regeneration-associated cells (RACs), which contain vasculogenic endothelial progenitor cells (EPCs) and anti-inflammatory/anti-immunity cells. Preclinical and clinical studies demonstrate that RAC transplantation is a safe and convenient cell population for promoting ischemic tissue recovery based on its strong vasculogenicity and functionality. The outputs of the scientific reports reviewed in the present study shed light on the fact that RAC transplantation is efficient in curing various diseases. Here, we compactly highlight the universal features of RACs and the latest progress in their translation toward clinics.

CD34 positive cells as endothelial progenitor cells in biology and medicine.

CD34 is a cell surface antigen expressed in numerous stem/progenitor cells including hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs), which are known to be rich sources of EPCs. Therefore, regenerative therapy using CD34+ cells has attracted interest for application in patients with various vascular, ischemic, and inflammatory diseases. CD34+ cells have recently been reported to improve therapeutic angiogenesis in a variety of diseases. Mechanistically, CD34+ cells are involved in both direct incorporation into the expanding vasculature and paracrine activity through angiogenesis, anti-inflammatory, immunomodulatory, and anti-apoptosis/fibrosis roles, which support the developing microvasculature. Preclinical, pilot, and clinical trials have well documented a track record of safety, practicality, and validity of CD34+ cell therapy in various diseases. However, the clinical application of CD34+ cell therapy has triggered scientific debates and controversies in last decade. This review covers all preexisting scientific literature and prepares an overview of the comprehensive biology of CD34+ cells as well as the preclinical/clinical details of CD34+ cell therapy for regenerative medicine.

Repetitive administration of cultured human CD34+ cells improve adenine-induced kidney injury in mice.

BACKGROUND: There is no established treatment to impede the progression or restore kidney function in human chronic kidney disease (CKD). AIM: To examine the efficacy of cultured human CD34+ cells with enhanced proliferating potential in kidney injury in mice. METHODS: Human umbilical cord blood (UCB)-derived CD34+ cells were incubated for one week in vasculogenic conditioning medium. Vasculogenic culture significantly increased the number of CD34+ cells and their ability to form endothelial progenitor cell colony-forming units. Adenine-induced tubulointerstitial injury of the kidney was induced in immunodeficient non-obese diabetic/severe combined immunodeficiency mice, and cultured human UCB-CD34+ cells were administered at a dose of 1 × 106/mouse on days 7, 14, and 21 after the start of adenine diet. RESULTS: Repetitive administration of cultured UCB-CD34+ cells significantly improved the time-course of kidney dysfunction in the cell therapy group compared with that in the control group. Both interstitial fibrosis and tubular damage were significantly reduced in the cell therapy group compared with those in the control group (P < 0.01). Microvasculature integrity was significantly preserved (P < 0.01) and macrophage infiltration into kidney tissue was dramatically decreased in the cell therapy group compared with those in the control group (P < 0.001). CONCLUSION: Early intervention using human cultured CD34+ cells significantly improved the progression of tubulointerstitial kidney injury. Repetitive administration of cultured human UCB-CD34+ cells significantly improved tubulointerstitial damage in adenine-induced kidney injury in mice via vasculoprotective and anti-inflammatory effects.

Inflammation in myocardial infarction: roles of mesenchymal stem cells and their secretome.

Inflammation plays crucial roles in the regulation of pathophysiological processes involved in injury, repair and remodeling of the infarcted heart; hence, it has become a promising target to improve the prognosis of myocardial infarction (MI). Mesenchymal stem cells (MSCs) serve as an effective and innovative treatment option for cardiac repair owing to their paracrine effects and immunomodulatory functions. In fact, transplanted MSCs have been shown to accumulate at injury sites of heart, exerting multiple effects including immunomodulation, regulating macrophages polarization, modulating the activation of T cells, NK cells and dendritic cells and alleviating pyroptosis of non-immune cells. Many studies also proved that preconditioning of MSCs can enhance their inflammation-regulatory effects. In this review, we provide an overview on the current understanding of the mechanisms on MSCs and their secretome regulating inflammation and immune cells after myocardial infarction and shed light on the applications of MSCs in the treatment of cardiac infarction.

Characterization of Endothelial Progenitor Cell: Past, Present, and Future.

Endothelial progenitor cells (EPCs) are currently being studied as candidate cell sources for revascularization strategies. Despite these promising results, widespread clinical acceptance of EPCs for clinical therapies remains hampered by several challenges. The challenges and issues surrounding the use of EPCs and the current paradigm being developed to improve the harvest efficiency and functionality of EPCs for application in regenerative medicine are discussed. It has been observed that controversies have emerged regarding the isolation techniques and classification and origin of EPCs. This manuscript attempts to highlight the concept of EPCs in a sequential manner, from the initial discovery to the present (origin, sources of EPCs, isolation, and identification techniques). Human and murine EPC marker diversity is also discussed. Additionally, this manuscript is aimed at summarizing our current and future prospects regarding the crosstalk of EPCs with the biology of hematopoietic cells and culture techniques in the context of regeneration-associated cells (RACs).

Transplantation of Fibroblast Sheets with Blood Mononuclear Cell Culture Exerts Cardioprotective Effects by Enhancing Anti-Inflammation and Vasculogenic Potential in Rat Experimental Autoimmune Myocarditis Model.

Fulminant myocarditis causes impaired cardiac function, leading to poor prognosis and heart failure. Cell sheet engineering is an effective therapeutic option for improving cardiac function. Naïve blood mononuclear cells (MNCs) have been previously shown to enhance the quality and quantity of cellular fractions (QQMNCs) with anti-inflammatory and vasculogenic potential using the one culture system. Herein, we investigated whether autologous cell sheet transplant with QQMNCs improves cardiac function in a rat model with experimental autoimmune myocarditis (EAM). Fibroblast sheets (F-sheet), prepared from EAM rats, were co-cultured with or without QQMNCs (QQ+F sheet) on temperature-responsive dishes. QQ+F sheet induced higher expression of anti-inflammatory and vasculogenic genes (Vegf-b, Hgf, Il-10, and Mrc1/Cd206) than the F sheet. EAM rats were transplanted with either QQ+F sheet or F-sheet, and the left ventricular (LV) hemodynamic analysis was performed using cardiac catheterization. Among the three groups (QQ+F sheet, F-sheet, operation control), the QQ+F sheet transplant group showed alleviation of end-diastolic pressure-volume relationship on a volume load to the same level as that in the healthy group. Histological analysis revealed that QQ+F sheet transplantation promoted revascularization and mitigated fibrosis by limiting LV remodeling. Therefore, autologous QQMNC-modified F-sheets may be a beneficial therapeutic option for EAM.

Personalized Cell Therapy for Patients with Peripheral Arterial Diseases in the Context of Genetic Alterations: Artificial Intelligence-Based Responder and Non-Responder Prediction.

Stem/progenitor cell transplantation is a potential novel therapeutic strategy to induce angiogenesis in ischemic tissue, which can prevent major amputation in patients with advanced peripheral artery disease (PAD). Thus, clinicians can use cell therapies worldwide to treat PAD. However, some cell therapy studies did not report beneficial outcomes. Clinical researchers have suggested that classical risk factors and comorbidities may adversely affect the efficacy of cell therapy. Some studies have indicated that the response to stem cell therapy varies among patients, even in those harboring limited risk factors. This suggests the role of undetermined risk factors, including genetic alterations, somatic mutations, and clonal hematopoiesis. Personalized stem cell-based therapy can be developed by analyzing individual risk factors. These approaches must consider several clinical biomarkers and perform studies (such as genome-wide association studies (GWAS)) on disease-related genetic traits and integrate the findings with those of transcriptome-wide association studies (TWAS) and whole-genome sequencing in PAD. Additional unbiased analyses with state-of-the-art computational methods, such as machine learning-based patient stratification, are suited for predictions in clinical investigations. The integration of these complex approaches into a unified analysis procedure for the identification of responders and non-responders before stem cell therapy, which can decrease treatment expenditure, is a major challenge for increasing the efficacy of therapies.

Extracellular Vesicles Derived From Regeneration Associated Cells Preserve Heart Function After Ischemia-Induced Injury.

Under vasculogenic conditioning, pro-inflammatory cell subsets of peripheral blood mononuclear cells (PBMCs) shift their phenotype to pro-regenerative cells such as vasculogenic endothelial progenitor cells, M2 macrophages, and regulatory T cells, collectively designated as regeneration-associated cells (RACs). In this study, we evaluated the therapeutic efficacy of RAC-derived extracellular vesicles (RACev) compared to mesenchymal stem cell-derived EVs (MSCev) in the context of myocardial ischemia reperfusion injury (M-IRI). Human PBMCs were cultured with defined growth factors for seven days to harvest RACs. RACev and MSCev were isolated via serial centrifugation and ultracentrifugation. EV quantity and size were characterized by nanoparticle tracking analysis. In vitro, RACev markedly enhanced the viability, and proliferation of human umbilical vein endothelial cells in a dose-dependent manner compared to MSCev. Notably, systemic injection of RACev improved cardiac functions at 4 weeks, such as fractional shortening, and protection from mitral regurgitation than the MSCev-treated group. Histologically, the RACev-transplanted group showed less interstitial fibrosis and enhanced capillary densities compared to the MSCev group. These beneficial effects were coupled with significant expression of angiogenesis, anti-fibrosis, anti-inflammatory, and cardiomyogenesis-related miRs in RACev, while modestly in MSCev. In vivo bioluminescence analysis showed preferential accumulation of RACev in the IR-injured myocardium, while MSCev accumulation was limited. Immune phenotyping analysis confirmed the immunomodulatory effect of MSCev and RACev. Overall, repetitive systemic transplantation of RACev is superior to MSCev in terms of cardiac function enhancements via crucial angiogenesis, anti-fibrosis, anti-inflammation miR delivery to the ischemic tissue.

Latest Advances in Endothelial Progenitor Cell-Derived Extracellular Vesicles Translation to the Clinic.

Almost all nucleated cells secrete extracellular vesicles (EVs) that are heterogeneous spheroid patterned or round shape particles ranging from 30 to 200 nm in size. Recent preclinical and clinical studies have shown that endothelial progenitor cell-derived EVs (EPC-EVs) have a beneficial therapeutic effect in various diseases, including cardiovascular diseases and kidney, and lung disorders. Moreover, some animal studies have shown that EPC-EVs selectively accumulate at the injury site with a specific mechanism of binding along with angiogenic and restorative effects that are superior to those of their ancestors. This review article highlights current advances in the biogenesis, delivery route, and long-term storage methods of EPC-EVs and their favorable effects such as anti-inflammatory, angiogenic, and tissue protection in various diseases. Finally, we review the possibility of therapeutic application of EPC-EVs in the clinic.

Acute kidney injury successfully treated with autologous granulocyte colony-stimulating factor-mobilized peripheral blood CD34-positive cell transplantation: A first-in-human report.

A 36-year-old man with severe acute kidney injury (AKI) was admitted to Shonan Kamakura General Hospital in Japan. He was diagnosed with refractory hypertension based on a severely elevated blood pressure of 224/116 mmHg and retinal, cardiac, and brain damage revealed by electrocardiogram, fundoscopy, and magnetic resonance imaging, respectively. Although hemodialysis was withdrawn following strict blood pressure control by an angiotensin receptor blocker, severe kidney insufficiency persisted. Therefore, we performed an autologous granulocyte colony-stimulating factor-mobilized peripheral blood CD34-positive cell transplantation. Collected CD34-positive cells were directly infused to both renal arteries. The patient's general condition was unremarkable after intervention, and the serum creatinine level gradually improved to 2.96 mg/dL 23 weeks after cell therapy. Although transient fever and thrombocytosis were observed after intervention, no major adverse events were observed. This patient is the first case in a phase I/II clinical trial of autologous granulocyte colony-stimulating factor-mobilized peripheral blood CD34-positive cell transplantation for severe AKI with a CD34-positive cell dose-escalating protocol (trial number jRCTb030190231).

Drinking Molecular Hydrogen Water Is Beneficial to Cardiovascular Function in Diet-Induced Obesity Mice.

Molecular hydrogen (MH) reportedly exerts therapeutic effects against inflammatory diseases as a suppressor of free radical chain reactions. Here, the cardiovascular protective effects of the intake of molecular hydrogen water (MHW) were investigated using high-fat diet-induced obesity (DIO) mice. MHW was prepared using supplier sticks and degassed water as control. MHW intake for 2 weeks did not improve blood sugar or body weight but decreased heart weight in DIO mice. Moreover, MHW intake improved cardiac hypertrophy, shortened the width of cardiomyocytes, dilated the capillaries and arterioles, activated myocardial eNOS-Ser-1177 phosphorylation, and restored left ventricular function in DIO mice. MHW intake promoted the histological conversion of hypertrophy to hyperplasia in white and brown adipose tissues (WAT and BAT) with the upregulation of thermogenic and cardiovascular protective genes in BAT (i.e., Ucp-1, Vegf-a, and eNos). Furthermore, the results of a colony formation assay of bone-marrow-derived endothelial progenitor cells (EPCs) indicated that MHW activated the expansion, differentiation, and mobilization of EPCs to maintain vascular homeostasis. These findings indicate that the intake of MHW exerts cardiovascular protective effects in DIO mice. Hence, drinking MHW is a potential prophylactic strategy against cardiovascular disorders in metabolic syndrome.

Ex vivo conditioning of peripheral blood mononuclear cells of diabetic patients promotes vasculogenic wound healing.

The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum-free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)-QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum-free expansion media with VEGF, Flt-3 ligand, TPO, IL-6, and SCF. The vascular regeneration capability of MNC-QQ cells pre- or post-QQc was evaluated with an EPC colony-forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 104 pre- and post-MNC-QQc cells from diabetic donors were injected into a murine wound-healing model using Balb/c nude mice. The percentage of wound closure and angio-vasculogenesis was then assessed. This study revealed vasculogenic, anti-inflammatory, and wound-healing effects of MNC-QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound-healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient-based vascular and regenerative therapy for patients with diabetes mellitus.

Hematopoietic stem-cell senescence and myocardial repair - Coronary artery disease genotype/phenotype analysis of post-MI myocardial regeneration response induced by CABG/CD133+ bone marrow hematopoietic stem cell treatment in RCT PERFECT Phase 3.

BACKGROUND: Bone marrow stem cell clonal dysfunction by somatic mutation is suspected to affect post-infarction myocardial regeneration after coronary bypass surgery (CABG). METHODS: Transcriptome and variant expression analysis was studied in the phase 3 PERFECT trial post myocardial infarction CABG and CD133+ bone marrow derived hematopoetic stem cells showing difference in left ventricular ejection fraction (∆LVEF) myocardial regeneration Responders (n=14; ∆LVEF +16% day 180/0) and Non-responders (n=9; ∆LVEF -1.1% day 180/0). Subsequently, the findings have been validated in an independent patient cohort (n=14) as well as in two preclinical mouse models investigating SH2B3/LNK antisense or knockout deficient conditions. FINDINGS: 1. Clinical: R differed from NR in a total of 161 genes in differential expression (n=23, q<0•05) and 872 genes in coexpression analysis (n=23, q<0•05). Machine Learning clustering analysis revealed distinct RvsNR preoperative gene-expression signatures in peripheral blood acorrelated to SH2B3 (p<0.05). Mutation analysis revealed increased specific variants in RvsNR. (R: 48 genes; NR: 224 genes). 2. Preclinical:SH2B3/LNK-silenced hematopoietic stem cell (HSC) clones displayed significant overgrowth of myeloid and immune cells in bone marrow, peripheral blood, and tissue at day 160 after competitive bone-marrow transplantation into mice. SH2B3/LNK-/- mice demonstrated enhanced cardiac repair through augmenting the kinetics of bone marrow-derived endothelial progenitor cells, increased capillary density in ischemic myocardium, and reduced left ventricular fibrosis with preserved cardiac function. 3. VALIDATION: Evaluation analysis in 14 additional patients revealed 85% RvsNR (12/14 patients) prediction accuracy for the identified biomarker signature. INTERPRETATION: Myocardial repair is affected by HSC gene response and somatic mutation. Machine Learning can be utilized to identify and predict pathological HSC response. FUNDING: German Ministry of Research and Education (BMBF): Reference and Translation Center for Cardiac Stem Cell Therapy - FKZ0312138A and FKZ031L0106C, German Ministry of Research and Education (BMBF): Collaborative research center - DFG:SFB738 and Center of Excellence - DFG:EC-REBIRTH), European Social Fonds: ESF/IV-WM-B34-0011/08, ESF/IV-WM-B34-0030/10, and Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany. Japanese Ministry of Health : Health and Labour Sciences Research Grant (H14-trans-001, H17-trans-002) TRIAL REGISTRATION: ClinicalTrials.gov NCT00950274.

Phase 1 clinical study of cell therapy with effective-mononuclear cells (E-MNC) for radiogenic xerostomia (first-in-human study) (FIH study on E-MNC therapy for radiogenic xerostomia).

BACKGROUND: Treatment for most patients with head and neck cancers includes ionizing radiation with or without chemotherapy. This treatment causes irreversible damage to salivary glands in the irradiation field accompanied by a loss of fluid-secreting acinar cells and a considerable decrease of saliva secretion. There is currently no adequate conventional treatment for this condition. In recent years, we developed an effective culture method to enhance the anti-inflammatory and vasculogenic phenotypes of peripheral blood mononuclear cells (PBMNCs), and such effectively conditioned PBMNC (E-MNC) therapy has shown promising improvements to the function of radiation-injured salivary glands in preclinical studies. However, the safety and effect of E-NMC therapy have yet assessed in human. The objective of this ongoing first-in-man study is to assess the safety, tolerability, and in part the efficacy of E-MNC therapy for treating radiation-induced xerostomia. METHODS/DESIGN: This phase 1 first-in-man study is an open-label, single-center, two-step dose escalation study. A total of 6 patients, who had no recurrence of head and neck cancer over 5 years following radiation therapy and suffered from radiation-induced xerostomia, will receive a transplantation of E-NMCs derived from autologous PBMNCs to a submandibular gland. The duration of the intervention will be 1 year. To analyze the recovery of salivary secretion, a gum test will be performed. To analyze the recovery of atrophic salivary glands, computed tomography (CT), and magnetic resonance imaging (MRI) of salivary glands will be conducted. The primary endpoint is the safety of the protocol. The secondary endpoints are the changes from baseline in whole saliva secretion and salivary gland atrophy. DISCUSSION: This will be the first clinical study of regenerative therapy using E-MNCs for patients with severe radiation-induced xerostomia. The results of this study are expected to contribute to developing the low-invasive cell-based therapy for radiation-induced xerostomia. TRIAL REGISTRATION: This study was registered with the Japan Registry of Clinical Trials (http://jrct.niph.go.jp) as jRCTb070190057.

Overview of Basic Mechanisms of Notch Signaling in Development and Disease.

Notch signaling is an evolutionarily conserved pathway associated with the development and differentiation of all metazoans. It is needed for proper germ layer formation and segmentation of the embryo and controls the timing and duration of differentiation events in a dynamic manner. Perturbations of Notch signaling result in blockades of developmental cascades, developmental anomalies, and cancers. An in-depth understanding of Notch signaling is thus required to comprehend the basis of development and cancer, and can be further exploited to understand and direct the outcomes of targeted cellular differentiation into desired cell types and complex tissues from pluripotent or adult stem and progenitor cells. In this chapter, we briefly summarize the molecular, evolutionary, and developmental basis of Notch signaling. We will focus on understanding the basics of Notch signaling and its signaling control mechanisms, its developmental outcomes and perturbations leading to developmental defects, as well as have a brief look at mutations of the Notch signaling pathway causing human hereditary disorders or cancers.

Author Correction: Insufficient production of IL-10 from M2 macrophages impairs in vitro endothelial progenitor cell differentiation in patients with Moyamoya disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Insufficient production of IL-10 from M2 macrophages impairs in vitro endothelial progenitor cell differentiation in patients with Moyamoya disease.

Moyamoya disease (MMD) is well known to be caused by insufficient cerebral vascular formation. However, the essential pathogenesis has not yet been identified. Using our recently developed technique of generating vasculogenic and anti-inflammatory cultures, we investigated endothelial progenitor cell (EPC) expansion and differentiation under the cytokine milieu generated by the peripheral blood mononuclear cells (PBMNCs) of the operated and non-operated MMD patients. EPC colony forming assay of the cultured PBMNCs disclosed the decline of the definitive EPC colony numbers in the both MMD patients. The level of interleukin-10 (IL-10) was lower in secretory cytokines from the cultured PBMNCs of MMD patients than that in that of controls using a cytometric bead array. The addition of human recombinant IL-10 to PBMNCs cultured from MMD patients restored the EPC colony forming potential of MMD PBMNCs. Following phorbol myristate acetate stimulation of the cultured PBMNCs, flow cytometry revealed a decrease in intracellular IL-10 storage in the main cell populations of the PBMNCs cultured from MMD patients relative to those cultured from controls. The present data provide the expected mechanism of vascular malformation in MMD pathogenesis originated from the insufficient production of IL-10 secreting cells from PBMNCs fostering EPC expansion and differentiation.

Anti-inflammatory and vasculogenic conditioning of peripheral blood mononuclear cells reinforces their therapeutic potential for radiation-injured salivary glands.

BACKGROUND: There are currently no effective treatments available for patients with irreversible loss of salivary gland (SG) function caused by radiation therapy for head and neck cancer. In this study, we have developed an effective culture method to enhance the anti-inflammatory and vasculogenic phenotypes of peripheral blood mononuclear cells (PBMNCs) and investigated whether such effectively conditioned PBMNCs (E-MNCs) could regenerate radiation-injured SGs and ameliorate salivary secretory function in mice. METHODS: Mouse PBMNCs were expanded in primary serum-free culture with five vasculogenic proteins for 5 days, and then the resulting cells (E-MNCs) were analyzed for their characteristics. Subsequently, 5 × 104 E-MNCs (labeled with EGFP in some experiments) were injected intra-glandularly into a mouse model of radiation-injured atrophic submandibular glands. After 2-3 weeks, the submandibular glands were harvested, and then the injected E-MNCs were tracked. Four, 8, and 12 weeks after irradiation (IR), salivary outputs were measured to evaluate the recovery of secretory function, and the gland tissues were harvested for histological and gene expression analyses to clarify the effects of cell transplantation. RESULTS: The resulting E-MNCs contained an enriched population of definitive CD11b/CD206-positive (M2 macrophage-like) cells and showed anti-inflammatory and vasculogenic characteristics. Salivary secretory function in E-MNC-transplanted mice gradually recovered after 4 weeks post-irradiation (post-IR) and reached 3.8-fold higher than that of non-transplanted mice at 12 weeks. EGFP-expressing E-MNCs were detected in a portion of the vascular endothelium and perivascular gland tissues at 2 weeks post-IR, but mainly in some microvessels at 3 weeks. Between 4 and 12 weeks post-IR, mRNA expression and histological analyses revealed that E-MNC transplantation reduced the expression of inflammatory genes and increased the level of tissue-regenerative activities such as stem cell markers, cell proliferation, and blood vessel formation. At 12 weeks post-IR, the areas of acinar and ductal cells regenerated, and the glands had less fibrosis. CONCLUSIONS: This effective conditioning of PBMNCs is a simple, rapid, and efficient method that provides a non-invasive source of therapeutic cells for regenerating radiation-injured atrophic SGs.

The Hedgehog Signaling Pathway in Ischemic Tissues.

Hedgehog (Hh) proteins are prototypical morphogens known to regulate epithelial/mesenchymal interactions during embryonic development. In addition to its pivotal role in embryogenesis, the Hh signaling pathway may be recapitulated in post-natal life in a number of physiological and pathological conditions, including ischemia. This review highlights the involvement of Hh signaling in ischemic tissue regeneration and angiogenesis, with particular attention to the heart, the brain, and the skeletal muscle. Updated information on the potential role of the Hh pathway as a therapeutic target in the ischemic condition is also presented.

Batroxobin accelerated tissue repair via neutrophil extracellular trap regulation and defibrinogenation in a murine ischemic hindlimb model.

Batroxobin, isolated from Bothrops moojeni, is a defibrinogenating agent used as a thrombin-like serine protease against fibrinogen for improving microcirculation. Here, we investigated whether, and if so, how batroxobin restores ischemic tissue injury in terms of anti-inflammatory effects. In an in vitro flow cytometry assay for human neutrophil extracellular traps (NETs), batroxobin (DF-521; Defibrase) inhibited human NETs induced by tumor necrosis factor-α (TNF-α) in the presence of human fibrinogen. Next, the effect of batroxobin was investigated by immunohistochemistry of the anterior tibial muscle (ATM) in an ischemic hindlimb model using C57BL/6J mice intraperitoneally injected with DF-521 versus the saline control. NETs and fibrinogen deposition in the ischemic ATM decreased in DF-521-treated mice on day 2 after ischemia. Meanwhile, reverse transcription-quantitative PCR assay of the ischemic ATM unveiled continuous downregulation in the expression of the genes; Tnf-α and nitric oxide synthase2 (Nos2) with hypoxia-inducible factor-1α (Hif-1α) and vascular endothelial growth factor-a (Vegf-a) from day 3 to day 7, but the upregulation of arginase-1 (Arg-1) and placental growth factor (Plgf) with myogenin (Myog) on day 7. Daily intraperitoneal DF-521 injection for the initial 7 days into mice with ischemic hindlimbs promoted angiogenesis and arteriogenesis on day 14. Moreover, DF-521 injection accelerated myofiber maturation after day 14. Laser doppler imaging analysis revealed that blood perfusion in DF-521-injected mice significantly improved on day 14 versus the saline control. Thus, DF-521 improves microcirculation by protecting NETs with tissue defibrinogenation, thereby protecting against severe ischemic tissue injury and accelerating vascular and skeletal muscular regeneration. To our knowledge, batroxobin might be the first clinically applicable NET inhibitor against ischemic diseases.