Development of strategies for vascular damage repair in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive and rare disease with so far unclear pathogenesis, limited treatment options and poor prognosis. Unbalance of proliferation and migration in pulmonary arterial smooth muscle cells (PASMCs) is an important hallmark of PAH. In this research Sodium butyrate (BU) has been evaluated in vitro and in vivo models of PAH. This histone deacetylase inhibitor (HDACi) counteracted platelet-derived growth factor (PDGF)-induced ki67 expression in PASMCs, and arrested cell cycle mainly at G0/G1 phases. Furthermore, BU reduced the transcription of PDGFRbeta, and that of Ednra and Ednrb, two major receptors in PAH progression. Wound healing and pulmonary artery ring assays indicated that BU inhibited PDGF-induced PASMC migration. BU strongly inhibited PDGF-induced Akt phosphorylation, an effect reversed by the phosphatase inhibitor calyculinA. In vivo, BU showed efficacy in monocrotaline-induced PAH in rats. Indeed, the HDACi reduced both thickness of distal pulmonary arteries and right ventricular hypertrophy. Besides these studies, Serial Analysis of Gene Expression (SAGE) has be used to obtain complete transcriptional profiles of peripheral blood mononuclear cells (PBMCs) isolated from PAH and Healthy subjects. SAGE allows quantitative analysis of thousands transcripts, relying on the principle that a short oligonucleotide (tag) can uniquely identify mRNA transcripts. Tag frequency reflects transcript abundance. We enrolled patients naïve for a specific PAH therapy (4 IPAH non-responder, 3 IPAH responder, 6 HeritablePAH), and 8 healthy subjects. Comparative analysis revealed that significant differential expression was only restricted to a hundred of down- or up-regulated genes. Interestingly, these genes can be clustered into functional networks, sharing a number of crucial features in cellular homeostasis and signaling. SAGE can provide affordable analysis of genes amenable for molecular dissection of PAH using PBMCs as a sentinel, surrogate tissue. Altogether, these findings may disclose novel perspectives in the use of HDACi in PAH and potential biomarkers.

Balloon pulmonary angioplasty in chronic thromboembolic pulmonary hypertension: experience of four years in a single centre

Background: Balloon pulmonary angioplasty (BPA) has recently been developed as an alternative and less- invasive treatment strategy for chronic thromboembolic pulmonary hypertension (CTEPH), but therapeutic efficacy and technical safety of the technique have to be established. Aim: effects of BPA on patients with inoperable disease or residual pulmonary hypertension (PH) after pulmonary endarterectomy (PEA). Methods: From June 2015 to September 2019 we enrolled symptomatic (NYHA ≥ II) inoperable CTEPH patients and patients with residual PH after PEA. At baseline, immediately before the first BPA session and 3-6 months after last BPA session all patients underwent clinical evaluation, six-minute walking distance and right heart catheterization. For comparisons Friedman test (with Bonferroni post-hoc pairwise analysis) was used. Survival curves were done with Kaplan Meier method. Results: Forty-seven patients [male 45%, median age 68 (51-74) years, 40 inoperable and 7 with residual PH after PEA] were treated for a total of 136 sessions (median number of sessions for each patient: 2); during each session we treated 2 (2-3) vessels; BPA significantly improved symptoms (NYHA III-IV from 85 to 42%), exercise capacity (from 425 to 446 m) and hemodynamic profile (reduction of mean pulmonary arterial pressure from 41 to 35 mmHg and of pulmonary vascular resistance from 7.1 to 4.7 WU). Five pulmonary artery dissection and 2 hemoptysis with clinical impairment were documented; 33 patients had lung injury (radiographic opacity with/without hemoptysis and/or hypoxemia), 7 patients had access site complications. Five patients died during follow-up (none within 30 days from the procedure) because of sepsis (1), heart failure (1), cancer (1), arrhythmic storm (1) and sudden death in a patient with severe coronary atherosclerosis (1). Conclusions: BPA is a safe and effective treatment able to improve symptoms and hemodynamic profile in inoperable CTEPH patients and in patients with residual PH after PEA.

Design and Synthesis of Novel Kinase Inhibitors for the Treatment of Chronic Respiratory Diseases

In 2017, Chronic Respiratory Diseases accounted for almost four million deaths worldwide. Unfortunately, current treatments are not definitive for such diseases. This unmet medical need forces the scientific community to increase efforts in the identification of new therapeutic solutions. PI3K delta plays a key role in mechanisms that promote airway chronic inflammation underlying Asthma and COPD. The first part of this project was dedicated to the identification of novel PI3K delta inhibitors. A first SAR expansion of a Hit, previously identified by a HTS campaign, was carried out. A library of 43 analogues was synthesised taking advantage of an efficient synthetic approach. This allowed the identification of an improved Hit of nanomolar enzymatic potency and moderate selectivity for PI3K delta over other PI3K isoforms. However, this compound exhibited low potency in cell-based assays. Low cellular potency was related to sub optimal phys-chem and ADME properties. The analysis of the X-ray crystal structure of this compound in human PI3K delta guided a second tailored SAR expansion that led to improved cellular potency and solubility. The second part of the thesis was focused on the rational design and synthesis of new macrocyclic Rho-associated protein kinases (ROCKs) inhibitors. Inhibition of these kinases has been associated with vasodilating effects. Therefore, ROCKs could represent attractive targets for the treatment of pulmonary arterial hypertension (PAH). Known ROCK inhibitors suffer from low selectivity across the kinome. The design of macrocyclic inhibitors was considered a promising strategy to obtain improved selectivity. Known inhibitors from literature were evaluated for opportunities of macrocyclization using a knowledge-based approach supported by Computer Aided Drug Design (CADD). The identification of a macrocyclic ROCK inhibitor with enzymatic activity in the low micro molar range against ROCK II represented a promising result that validated this innovative approach in the design of new ROCKs inhibitors.

Psychological characterization of hypertensive patients: associations with adherence to pharmacological treatment and self-management

Introduction: The role of psychosocial factors in the onset and progression of essential hypertension has been object of a large body of literature, yet findings appear to be controversial. Aims: We assessed the predictive role of psychosomatic syndromes, affective symptomatology, psychological reactance, psychological distress, well-being and quality of life on adherence to antihypertensive medications, lifestyle behaviors, hypertension severity and absolute cardiovascular risk grading, as well as their temporal stability at 1-year follow-up, in a sample of hypertensive patients. In addition, we aimed to validate the Italian version of the Hong Psychological Reactance Scale (HPRS). Methods: Eighty consecutive hypertensive outpatients treated with antihypertensive medications were compared to 80 controls. Psychosocial variables were assessed using clinical interviews and self-rating questionnaires at baseline and at 1-year follow-up. Cardiac parameters were also collected. One-hundred and fifty individuals from general population provided data for the HPRS validation. Results: Hypertensive patients reported significantly higher levels of psychological distress and lower levels of psychological well-being at baseline compared to controls. Among hypertensive patients, allostatic overload (AO) was the most frequently reported psychosomatic syndrome at baseline. Patients with AO displayed significantly greater levels of psychological distress and lower levels of well-being and quality of life than those without. Further, patients with illness denial were significantly more likely to report poor adherence to pharmacological treatment and, as well as those with higher levels of affective symptomatology, were less likely to follow a balanced diet. At follow-up, patients displayed significantly higher levels of well-being and lower levels of stress, mental pain and quality of life. Conclusions: Findings suggest the clinical relevance of psychosocial factors and psychosomatic syndromes in the progression of hypertension, with important implications for its management. As to the Italian validation of the HPRS, results support previous findings, even though a confirmatory factor analysis should be carried out.

Triple combination therapy in pulmonary arterial hypertension

Pulmonary arterial hypertension is a severe disease characterized by increasing in pulmonary vascular resistance leading to right ventricular failure and death. Currently available drugs for treatment of PAH act on three different pathways responsible of the pathogenesis of this disease: the endothelin pathway, the nitric oxide pathway and the prostacyclin pathway. The purpose of our study was to reassess our experience on the use of drugs that interact on the pathobiological line of prostacyclin so we retrospectively included all patients, referred to our center from February 1995 to December 2021, who received therapy with i.v. Epoprostenol, s.c. Treprostinil or oral Selexipag. Firstly, we observed that patients treated with Epoprostenol were significantly more compromised at baseline when compared to the two other groups and evaluating the effects of the three different drugs, it emerged that patients treated with Epoprostenol had significantly greater improvements in respect to those treated with Treprostinil and Selexipag. In the second part of our analysis we assessed the effects of these drugs when used as third line strategy in order to limit many confounding factors that could influence demographic, clinical and hemodynamic characteristics of patient populations. The differences emerged in exercise capacity and baseline hemodynamics reflect the fact that in our clinical practice, we add Epoprostenol as third line therapy in more compromised patients, Treprostinil in intermediate situations and Selexipag in less impaired conditions. Comparing the effects of treatments between baseline and first follow-up we noticed smaller benefits with Selexipag when compared with intravenous and subcutaneous strategies but it’s important to weight baseline patient’s differences. Our analysis confirmed clinical and functional benefits for the use of both prostacyclin analogues and prostacyclin receptor agonists in terms of improved functional class, six-minute walking distance and cardiopulmonary hemodynamics.