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Background Fibrosis and vascular disease are cardinal features of systemic sclerosis (SSc). Stimulators of soluble guanylate cyclase (sGC) are vasoactive drugs that are currently being evaluated in phase III clinical trials for pulmonary arterial hypertension. ObjectiveTo study the antifibrotic potency of sGC stimulators. Methods The effect of the sGC stimulator BAY 41-2272 on the release of collagen from dermal fibroblasts was examined. The antifibrotic effects of BAY 41-2272 on prevention and regression of fibrosis in bleomycin-induced dermal fibrosis and in Tsk-1 mice were also studied. Telemetric blood pressure studies in conscious mice were used to study potential hypotensive effects of sGC stimulation. Results sGC stimulation with BAY 41-2272 dose-dependently inhibited collagen release in dermal fibroblasts from patients with SSc and healthy individuals. Furthermore, BAY 41-2272 stopped the development of bleomycin-induced dermal fibrosis and skin fibrosis in Tsk-1 mice, preventing dermal and hypodermal thickening, reducing the numbers of myofibroblasts and reducing the hydroxyproline content. In addition, BAY 41-2272 was highly effective in the treatment of established fibrosis in the modified models of bleomycin-induced skin fibrosis and Tsk-1 mice. Treatment with sGC stimulators was well tolerated. Relevant antifibrotic doses of BAY 41-2272 did not affect systemic blood pressure and heart rate in mice. Conclusions These findings demonstrate potent antifibrotic effects and good tolerability of sGC stimulators in various experimental models of SSc. Given their potential vasoactive properties, sGC stimulators may be promising candidates for the dual treatment of fibrosis and vascular disease in SSc.

We have generated a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These 'smart' probes emit a fluorescent signal only after covalently modifying a specific protease target. After intravenous injection of NIRF-ABPs into mice bearing grafted tumors, noninvasive, whole-body imaging allowed direct monitoring of cathepsin activity. Importantly, the permanent nature of the probes also allowed secondary, ex vivo biochemical profiling to identify specific proteases and to correlate their activity with whole-body images. Finally, we demonstrate that these probes can be used to monitor small-molecule inhibition of protease targets both biochemically and by direct imaging methods. Thus, NIRF-ABPs are (i) potentially valuable new imaging agents for disease diagnosis and (ii) powerful tools for preclinical and clinical testing of small-molecule therapeutic agents in vivo .

IntroductionHypertension is a major cause of premature death worldwide as it is an important risk factor for coronary artery disease, myocardial infarction, heart failure and stroke. Although an estimated 1.3 billion adults suffer from hypertension, less than half of them are diagnosed correctly and therefore receive sufficient treatment. Furthermore, only one fifth of those treated reach the therapy target of normotension. This significant deficit underlines the need for new therapy concepts to improve long-term health outcomes. Several studies have shown positive effects of digital health programmes in the disease management of ambulatory, long-term hypertension treatment. More research is needed to explore the abilities of digital health programmes as an innovative pathway in ambulatory healthcare.The eXPLORE study aims to evaluate the feasibility of a clinical trial on the impact of a supplementary digital therapy programme for the treatment of primary arterial hypertension.Methods and analysesThe eXPLORE study collects data in the setting of a prospective randomised controlled trial to evaluate methodological feasibility for larger-scaled follow-up research. The study compares a digital therapy programme using a smartphone application that is based on functions and algorithms creating tasks and recommendations based on individual health data to standard care for the treatment of primary arterial hypertension. The study period is 180 days, with a 90-day in-life phase followed by a 90-day follow-up phase. Baseline and follow-up data (3 months, 6 months follow-up) of all participants included is collected via questionnaire surveys as well as self-administered blood pressure monitoring. Patient inclusion, initial data acquisition and follow-up were carried out in an innovative remote setting. The study was initiated in November 2022 and is currently ongoing. Study outcome measures are changes in mean blood pressure, health literacy and self-sufficient health behaviour.Ethics and disseminationThe eXPLORE study is carried out in accordance with all applicable legal regulations. Cost-effectiveness is assured by continuous evaluation and documentation over the course of the study. All health-relevant data from the eXPLORE study will be provided for analyses and publication to the investigators of LMU Hospital. The study was approved by the local ethics committee of LMU Munich (project nr.: 22–0115).Trial registrationNCT05580068. Protocol Version: 1.5, 28.08.2023.

Soluble receptors to vascular endothelial growth factor (VEGF) can inhibit its angiogenic effect. Since angiogenesis is involved in wound repair, we hypothesized that adenovirus-mediated gene transfer of a soluble form of VEGF receptor 2 (Flk-1) would attenuate wound healing in mice. C57Bl/6J and genetically diabetic (db/db) mice (each n=20) received intravenous (i.v.) injections of recombinant adenoviruses (10 9 PFU) encoding the ligand-binding ectodomain of VEGF receptor 2 (Flk-1) or cDNA encoding the murine IgG2α Fc fragment (each n=10). At 4 days after gene transfer, two full-thickness skin wounds (0.8 cm) were created on the dorsum of each animal. Wound closure was measured over 9–14 days after which wounds were resected for histological analysis. Prior to killing, fluorescent microspheres were systemically injected for quantitation of wound vascularity. Single i.v. injections of adenoviruses encoding soluble Flk-1 significantly decreased wound angiogenesis in both wild-type and diabetic mice. Fluorescence microscopy revealed a 2.0-fold (wild type) and 2.9-fold (diabetic) reduction in wound vascularity in Flk-1-treated animals (p<0.05). Impairment of angiogenesis was confirmed by CD31 immunohistochemistry. Interestingly, despite significant reductions in wound vascularity, wound closure was not grossly delayed. Our data indicates that while VEGF function is essential for optimal wound angiogenesis, it is not required for wound closure.

Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor–like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension. Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps) reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs). Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC) were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot). In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs). Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1). These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.

Therapeutic angiogenesis aims at generating new blood vessels by delivering growth factors such as VEGF and FGF. Clinical trials are underway in patients with peripheral vascular and coronary heart disease. However, increasing evidence indicates that the new vasculature needs to be stabilized to avoid deleterious effects such as edema and hemangioma formation. Moreover, a major challenge is to induce new vessels that persist following cessation of the angiogenic stimulus. Mature vessels may be generated by modulating timing and dosage of growth factor expression, or by combination of ‘growth’ factors with ‘maturation’ factors like PDGF‐BB, angiopoietin‐1 or TGF‐β. Myoblast‐mediated gene transfer has unique characteristics that make it a useful tool for studying promising novel approaches to therapeutic angiogenesis. It affords robust and long‐lasting expression, and can be considered as a relatively rapid form of ‘adult transgenesis’ in muscle. The combined insertion of different gene constructs into single myoblasts and their progeny allows the simultaneous expression of different ‘growth’ and ‘maturation’ factors within the same cell in vivo. The additional insertion of a reporter gene makes it possible to analyze the phenotype of the vessels surrounding the transgenic muscle fibers into which the myoblasts have fused. The effects of timing and duration of gene expression can be studied by using tetracycline‐inducible constructs, and dosage effects by selecting subpopulations consistently expressing distinct levels of growth factors. Finally, the autologous cell‐based approach using transduced myoblasts could be an alternative gene delivery system for therapeutic angiogenesis in patients, avoiding the toxicities seen with some viral vectors. British Journal of Pharmacology (2003) 140, 620–626. doi:10.1038/sj.bjp.0705492

We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of β-galactosidase (β-gal) activity. The substrate, a caged D -luciferin–galactoside conjugate, must first be cleaved by β-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on β-gal activity. Using this technology, constitutive β-gal activity in engineered cells and inducible tissue-specific β-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of β-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant β-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo . Thus, coupling the properties of FLuc to the advantages of β-gal permits bioluminescent imaging applications that previously were not possible.

Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor-like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of beta-galactosidase (beta-gal) activity. The substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by beta-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered cells and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of beta-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant beta-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, coupling the properties of FLuc to the advantages of beta-gal permits bioluminescent imaging applications that previously were not possible.