Biology subjects

Madeddu, P.

Publications and source records attributed to Madeddu, P..

8 recordsLinked to original sources

bioRxiv2025

Molecular reprogramming of adventitial pericytes by a selective MEK inhibitor halts the progression of thoracic aortic aneurysm

AimReconstructive surgery is a life-saving treatment for individuals with advanced thoracic aortic aneurysms (TAAs) at risk of rupture. No effective pharmacological treatments are available to halt aortic dilatation before it reaches this critical point. Unravelling the cellular and molecular pathways involved in TAA formation and expansion is fundamental for identifying potential treatment targets. This study challenged the hypothesis that pericyte dysfunction destabilizes the adventitial vascular niche, hence compromising the ascending aortas elastic characteristics. We also investigated whether blocking the mitogen-activated extracellular signal-regulated kinase (MEK) pathway could slow TAA progression in a mouse model. MethodologyComparative histology and morphometry studies were performed on human ascending TAA and non-aneurismatic control tissues to quantify adventitia vasa vasorum (VV) size and abundance, and pericyte coverage and density. Proliferation, migration, and angiogenesis experiments were used to evaluate the functional phenotype of pericytes before and after blocking the MEK signalling pathway with PD0325901. In a mouse model with moderate TAA, we investigated the therapeutic efficacy of PD0325901 (10 mg/kg/d orally for 14 days) on TAA progression. ResultsThe histological study of TAA samples demonstrated VV remodelling and reduced pericyte VV coverage due to increased detachment. Cultured TAA pericytes exhibited aberrant behaviour, including increased proliferation, migration, matrix metalloprotease activity, disrupted angiogenic capacity, and altered secretome, and MEK overactivation. PD0325901 restored pericyte contractile phenotype and angiogenic capacity, influencing their secretome, migratory capacity, and matrix formation/degradation equilibrium. In vivo, PD0325901 remarkably decreased aortic dilatation, increased compliance, retained medial elastin content, and reduced adventitial inflammation. No harmful consequences were noted. ConclusionThis work identifies pericyte dysfunction related to MEK overactivation as a major contributor to TAA progression. This suggests that inhibiting the MEK signalling pathway could be a potential treatment option for TAA before surgical intervention becomes necessary. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIThoracic aortic aneurysm (TAA) remains a major clinical challenge due to its asymptomatic progression and risk of life-threatening rupture. Our study highlights the crucial role of adventitial pericytes in maintaining vascular homeostasis and preventing aneurysm-related vascular remodeling. C_LIO_LIWe demonstrate that dysregulated MEK/ERK signaling drives aortic adventitial pericyte dysfunction, leading to microvascular instability, extracellular matrix degradation, chronic inflammation, and progressive aneurysm expansion. C_LIO_LIImportantly, we show that the clinically available MEK inhibitor, PD0325901, effectively restores pericyte function, preserving adventitial vascular integrity, reducing inflammatory cytokine production, and stabilizing the aortic wall. C_LIO_LIIn a preclinical mouse model, PD0325901 significantly attenuated aneurysm growth, improved aortic wall compliance, and prevented maladaptive vascular remodeling. C_LI What Are the Clinical Implications?O_LIOur findings provide strong translational evidence supporting MEK inhibition as a promising therapeutic strategy to halt TAA progression and enhance aortic wall resilience, offering a potential medical alternative to delay or prevent surgical intervention. C_LI

cell biology↗

bioRxiv2023

Inhibition of MMP 2 protects the endothelial glycocalyx and improves diastolic function in diabetic cardiomyopathy

The coronary microvascular endothelial glycocalyx (EGlx) is a vital regulator of vascular permeability and EGlx damage contributes to the development of diabetic cardiomyopathy. Matrix metalloproteinases 2 and 9 (MMP2/9) have been identified as key enzymes in the degradation of EGlx components, notably syndecan 4 (SDC4), and are upregulated in diabetes. We tested the hypothesis that inhibition of MMP2/9 can protect the EGlx and improve diastolic function in diabetic cardiomyopathy. Type 1 diabetes was induced in FVB mice by streptozotocin (STZ) injections. Mice were treated with daily injections of the MMP2/9 inhibitor, SB-3CT, for 2 weeks from 7 weeks post STZ. Echocardiography was utilised to assess heart function and lectin staining for the measurement of EGlx depth. Immunolabelling of heart sections for albumin provided an indication of albumin extravasation. A mechanism of EGlx shedding was investigated in vitro in human coronary microvascular endothelial cells treated with TNF- and SB-3CT. Diabetic mice developed diastolic dysfunction from 6 weeks post STZ. MMP2/9 inhibition reversed diastolic dysfunction, EGlx thinning and albumin extravasation in diabetic animals. In vitro, TNF- caused an increase in MMP9 activity and SDC4 shedding from human coronary microvascular endothelial cells. Treatment with SB-3CT reduced MMP9 activity and prevented SDC4 shedding. Knockdown of MMP9 expression prevented TNF- induced SDC4 shedding. This study demonstrates MMP2/9 inhibition as a strategy to protect the EGlx and improve diastolic function in diabetic cardiomyopathy. Our findings suggest new avenues for therapeutic interventions in cardiovascular complications associated with diabetes. Statements and DeclarationsO_ST_ABSCompeting interestsC_ST_ABSThe authors have no competing interests to declare that are relevant to the content of this article.

physiology↗

bioRxiv2023

Regulation of BACH1 by hemin improves cardiac function in a mouse model of myocardial infarction

AimsThe BTB and CNC homology 1 (BACH1) transcription factor is a repressor of heme oxygenase-1 (HMOX1), a pivotal enzyme involved in antioxidant response and iron recycling. Here we investigated whether pharmacological modulation of the BACH1 by hemin impacts on antioxidant responses and reparative angiogenesis in a mouse model of myocardial infarction (MI). Methods and resultsIn vitro studies on vascular cells showed hemin treatment downregulates BACH1 gene and protein expression and upregulates HMOX1. This axis was confirmed to be modulated in the murine infarcted heart, with BACH1 being upregulated, and HMOX1 downregulated compared to sham. Treatment with hemin every 3 days for 28 days post-MI significantly decreased BACH1 and increased HMOX1 protein expression, though no decrease in oxidative stress markers was detected. Hemin treated mice showed increases in both capillary and arteriole density, and reduced iron accumulation compared with controls. Furthermore, echocardiology measurements showed hemin treatment induced significant improvements in left ventricular wall thickness, and cardiac function as indicated by increased ejection fraction, fractional shortening, and stroke volume measurements. ConclusionHemin has therapeutic potential to improve revascularisation and cardiac function in the heart post-MI.

molecular biology↗

bioRxiv2022

EZH2 interacts with MEG3 to directly regulate integrin signaling and endothelial cell function

Enhancer of Zeste Homologue 2 (EZH2) modulates gene transcription during endothelial cell (EC) dysfunction, via interaction with non-coding RNAs (ncRNAs). Thus, EZH2 can act as a rheostat in deposition of histone H3K27 trimethylation (H3K27me3) to repress many genes. We profiled EZH2-RNA interactions using formaldehyde/UV assisted cross-linking ligation and sequencing of hybrids (FLASH-seq) in primary human ECs. Transcriptome-wide EZH2-associated ncRNAs and RNA-RNA interactome were obtained. This approach revealed EZH2 directly binding maternally expressed gene (MEG3) and MEG3:MEG3 hybrid structures. By chromatin immunoprecipitation with sequencing (ChIP-seq) following depletion of MEG3, we discovered that MEG3 targets and controls recruitment of EZH2/H3K27me3 onto a regulatory region of integrin subunit alpha 4 (ITGA4). MEG3 knockdown or pharmacological inhibition of EZH2 de-repressed ITGA4, whilst improving endothelial cell function in vitro, and increasing ITGA4 expression in vivo. Our study demonstrates new role for MEG3, as instrumental in epigenetic regulation of EC function by EZH2, through targeting of integrin-dependent signalling.

molecular biology↗

bioRxiv2020

The SARS-CoV-2 spike protein disrupts the cooperative function of human cardiac pericytes - endothelial cells through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease

Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known whether heart vascular pericytes (PCs) are infected by SARS-CoV-2, and if the S protein alone provokes PC dysfunction. Here, we aimed to investigate the effects of the S protein on primary human cardiac PC signalling and function. Results show, for the first time, that cardiac PCs are not permissive to SARS-CoV-2 infection in vitro, whilst a recombinant S protein alone elicits functional alterations in PCs. This was documented as: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors responsible for EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation and rescued PC function in the presence of the S protein. In conclusion, our findings suggest that circulating S protein prompts vascular PC dysfunction, potentially contributing to establishing microvascular injury in organs distant from the site of infection. This mechanism may have clinical and therapeutic implications. Clinical perspectiveO_LISevere COVID-19 manifests as a microvascular syndrome, but whether SARS-CoV-2 infects and damages heart vascular pericytes (PCs) remains unknown. C_LIO_LIWe provide evidence that cardiac PCs are not infected by SARS-CoV-2. Importantly, we show that the recombinant S protein alone elicits cellular signalling through the CD147 receptor in cardiac PCs, thereby inducing cell dysfunction and microvascular disruption in vitro. C_LIO_LIThis study suggests that soluble S protein can potentially propagate damage to organs distant from sites of infection, promoting microvascular injury. Blocking the CD147 receptor in patients may help protect the vasculature not only from infection, but also from the collateral damage caused by the S protein. C_LI

cell biology↗

bioRxiv2020

Contractile reprogramming of cardiac pericytes by MEK inhibition promotes arteriologenesis of the ischemic heart

BackgroundThe development of collateral arteries after a myocardial infarction (MI) was intensively studied, while the mechanism by which pericytes (PCs) contribute to arteriologenesis remains unexplored. This study aimed to 1) investigate if cardiac PCs gain functional features of contractile vascular smooth muscle cells (VSMCs) in vitro, and 2) determine if this potential can be evoked pharmacologically to encourage heart arteriologenesis in vivo. MethodsPCs were immunosorted as CD31neg/CD34pos cells from human and mouse hearts. Contractile reprogramming was induced by either depletion of growth factors or addition of PD0325901, a clinically available MEK inhibitor. Next generation RNA-Sequencing was performed in naive and differentiated human PCs to assess the whole-transcriptome profile. Three in vivo studies were conducted in C57BL6/J mice to determine: 1) the ability of human PCs to promote arteriole formation when implanted subcutaneously within PD0325901-containing Matrigel plugs, 2) the effect of orally administered PD0325901 on the arteriole density of normoperfused hearts, and 3) the possibility of promoting capillary formation and muscularization of the infarcted heart through the same pharmacological approach. ResultsRemoval of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) from the culture medium induced the differentiation of PCs into contractile VSMC-like cells. Because both growth factors induce the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling, we attempted to induce PC differentiation in vitro and in vivo using PD0325901. RNA-sequencing revealed that differentiated PCs were enriched in transcripts associated with smooth muscle contraction and biological function. PD0325901-treated PCs rapidly acquired antigenic and functional features of contractile VSMCs in vitro. Moreover, human PCs formed new arterioles when implanted subcutaneously within PD0325901-containing Matrigel plugs in mice. Oral administration of PD0325901 for two weeks increased the density and expression of contractile proteins in small-calibre arterioles of the murine heart, thereby increasing myocardial perfusion. Similarly, PD0325901 induced reparative arteriologenesis and capillarization, reduced the scar, and improved left ventricular performance in a murine model of MI. ConclusionWe propose a novel method to promote the heart vascularization through the pharmacological modulation of resident mural cells. This novel approach could have an immediate impact on the treatment of coronary artery disease. Clinical perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIHuman myocardial pericytes have intrinsic vascular plasticity that can be pharmacologically evoked using PD0325901, a clinically available MEK inhibitor. C_LIO_LIIn mice, the pharmacological inhibition of ERK1/2 signalling, by the oral administration of PD0325901 for 2 weeks, encouraged the heart arteriologenesis through pericyte differentiation. C_LIO_LIIn a preclinical mouse model of myocardial infarction, the oral administration of PD0325901 for 2 weeks induced reparative arteriologenesis and capillarization, reduced the scar, and improved left ventricular performance. C_LI What are the clinical implications?O_LIThis novel drug-based therapeutic approach is readily available to all patients. C_LIO_LITherefore, it could have an immediate clinical impact for the treatment of coronary artery disease and other heart conditions associated with deficient coronary vascularization. C_LI

cell biology↗

bioRxiv2020

Human saphenous vein provides a unique source of anti-calcific pericytes for prosthetic cardiac valve engineering

AimsTissue engineering seeks to improve the longevity of prosthetic heart valves, but the cell source of choice has yet to be determined. This study aimed to establish a mechanistic rationale supporting the suitability of human adventitial pericytes (APCs). Methods and ResultsAntigenically APCs were immunomagnetically sorted from saphenous vein leftovers of patients undergoing coronary artery surgery and antigenically characterized for purity. Unlike bone marrow-derived mesenchymal stromal cells (BM-MSCs), APCs were resistant to osteochondrogenic induction by high phosphate (HP), as assessed by cytochemistry and expression of osteogenic markers. MiR-132 is natively expressed by APCs, with copy numbers being enhanced by HP stimulation. In silico bioinformatic analysis, followed by luciferase assays in HEK293 cells and miR-132 titration using agomiR and antagomiR in APCs, demonstrated that several osteochondrogenic genes were negatively regulated by miR-132. Among these, the glycolytic marker GLUT1 was downregulated in HP-stimulated APCs. In contrast to BM-APCs, APCs showed no increase in glycolysis under HP. Interestingly, incubation with APC-derived conditioned medium conferred swine cardiac valves with resistance to osteogenic transformation by HP; whereas, conditioned media from miR-132-knocked-down APCs failed to prevent the expression of these markers. Finally, we demonstrated the feasibility of using APCs to engineer bovine pericardium patches. APCs proliferate in the patch and secrete factors able to attract aortic endothelial cells under HP. ConclusionsHuman APCs are resistant to calcification compared with BM-MSCs and convey the anti-calcific phenotype to heart valves through miR-132. These findings may open new important avenues for prosthetic valve cellularization.

cell biology↗

bioRxiv2020

A Critical Role for Perivascular Cells in Amplifying Viral Haemorrhage Induced by Dengue Virus Non-Structural Protein 1

Dengue is the most prevalent arthropod-borne viral disease affecting humans, with severe dengue typified by potentially fatal microvascular leakage and hypovolaemic shock. Blood vessels of the microvasculature are composed of a tubular structure of endothelial cells ensheathed by perivascular cells (pericytes). Pericytes support endothelial cell barrier formation and maintenance through paracrine and contact-mediated signalling, and are critical to microvascular integrity. Pericyte dysfunction has been linked to vascular leakage in noncommunicable pathologies such as diabetic retinopathy, but has never been linked to infection-related vascular leakage. Dengue vascular leakage has been shown to result in part from the direct action of the secreted dengue virus (DENV) non-structural protein NS1 on endothelial cells. Using primary human vascular cells, we show here that NS1 also causes pericyte dysfunction, and that NS1-induced endothelial hyperpermeability is more pronounced in the presence of pericytes. Notably, NS1 specifically disrupted the ability of pericytes to support endothelial cell function in a 3D microvascular assay, with no effect on pericyte viability or physiology. These effects are mediated at least in part through contact-independent paracrine signals involved in endothelial barrier maintenance by pericytes. We therefore identify a role for pericytes in amplifying NS1-induced microvascular hyperpermeability in severe dengue, and thus show that pericytes can play a critical role in the aetiology of an infectious vascular leakage syndrome. These findings open new avenues of research for the development of drugs and diagnostic assays for combating infection-induced vascular leakage, such as severe dengue. SIGNIFICANCE STATEMENTThe World Health Organisation considers dengue one of the top ten global public health problems. There is no specific antiviral therapy to treat dengue virus and no way of predicting which patients will develop potentially fatal severe dengue, typified by vascular leakage and circulatory shock. We show here that perivascular cells (pericytes) amplify the vascular leakage-inducing effects of the dengue viral protein NS1 through contact-independent signalling to endothelial cells. While pericytes are known to contribute to noncommunicable vascular leakage, this is the first time these cells have been implicated in the vascular effects of an infectious disease. Our findings could pave the way for new therapies and diagnostics to combat dengue, and potentially other infectious vascular leakage syndromes.

microbiology↗