Intercellular adhesion plays a major role in tissue development and homeostasis. Yet, technologies to measure mature cell-cell contacts are not available. We introduce a methodology based on fluidic probe force microscopy to assess cell-cell adhesion forces after formation of mature intercellular contacts in cell monolayers. With this method we quantify that L929 fibroblasts exhibit negligible cell-cell adhesion in monolayers whereas human endothelial cells from the umbilical artery (HUAECs) exert strong intercellular adhesion forces per cell. We use a new in vitro model based on the overexpression of Muscle Segment Homeobox 1 (MSX1) to induce Endothelial-to-Mesenchymal Transition (EndMT), a process involved in cardiovascular development and disease. We reveal how intercellular adhesion forces in monolayer decrease significantly at an early stage of EndMT and we show that cells undergo stiffening and flattening at this stage. This new biomechanical insight complements and expands the established standard biomolecular analyses. Our study thus introduces a novel tool for the assessment of mature intercellular adhesion forces in a physiological setting that will be of relevance to biological processes in developmental biology, tissue regeneration and diseases like cancer and fibrosis.

We established an inverse correlation between endothelial PEAR1 expression and vascular assembly both in vitro and in vivo. These findings identify PEAR1 as a novel modifier of neoangiogenesis.

During peripheral arterial disease, MSX1 acts downstream of BMP–SMAD signaling to transduce the arterial shear stimulus into an arteriogenic remodeling response. MSX1 activates collateral endothelium into a proinflammatory state through ICAM1/VCAM1 up-regulation, resulting in increased leukocyte infiltration and collateral remodeling.

Rationale: Understanding the mechanisms that regulate arterial flow recovery is important to design treatment options for peripheral arterial disease (PAD) patients ineligible for invasive revascularization. Transcriptional orchestrators of this recovery process represent an appealing target for treatment design. We previously identified positive regulatory domain-containing protein (Prdm)16 as an arterial-specific endothelial transcription factor but its in vivo role in arteries remains completely unknown. Objective: To unravel the role of Prdm16 in arteries under physiological and pathological conditions, more specifically during PAD. Methods and Results: Methods and Results: Within the vasculature, Prdm16 expression was strictly expressed by arterial endothelial and smooth muscle cells. Heterozygous loss of Prdm16 caused a modest reduction of the inner arterial diameter and smooth muscle cell coating without compromising vasomotor function. Upon femoral artery ligation, Prdm16 +/- mice featured significantly impaired flow recovery to ischemic limbs. This impairment was recapitulated in mice with a Prdm16 deletion specifically in endothelial cells (EC-Prdm16 -/- ) but not smooth muscle cells. Structural ollateral remodeling was normal in both Prdm16 +/- and <EC-Prdm16 -/- mice, but significant endothelial dysfunction post-ligation was present in EC-Prdm16 -/- mice as evidenced by impaired endothelial-dependent relaxation. Upon ligation, endothelial Prdm16 deficiency altered the expression of genes encoding endothelial cell function regulators, many related to nitric oxide bioavailability and Ca2 + homeostasis. Accordingly, Prdm16 overexpression in cultured endothelial cells affected both total cellular Ca2 + levels and store-operated Ca2 + entry. Conclusions: Conclusions: We showed that Prdm16 is indispensable for arterial flow recovery under pathological challenge not because it affects structural remodeling but due to its role in maintaining endothelial function. It therefore represents an appealing target for designing novel therapeutic strategies for no-option patients with PAD.

Background: Chronic pressure overload predisposes to heart failure, but the pathogenic role of microvascular endothelial cells (MiVEC) remains unknown. We characterized transcriptional, metabolic, and functional adaptation of cardiac MiVEC to pressure overload in mice and patients with aortic stenosis (AS). Methods: In Tie2-Gfp mice subjected to transverse aortic constriction or sham surgery, we performed RNA sequencing of isolated cardiac Gfp + -MiVEC and validated the signature in freshly isolated MiVEC from left ventricle outflow tract and right atrium of patients with AS. We next compared their angiogenic and metabolic profiles and finally correlated molecular and pathological signatures with clinical phenotypes of 42 patients with AS (50% women). Results: In mice, transverse aortic constriction induced progressive systolic dysfunction, fibrosis, and reduced microvascular density. After 10 weeks, 25 genes predominantly involved in matrix-regulation were >2-fold upregulated in isolated MiVEC. Increased transcript levels of Cartilage Intermediate Layer Protein ( Cilp ), Thrombospondin-4 , Adamtsl-2 , and Collagen1a1 were confirmed by quantitative reverse transcription polymerase chain reaction and recapitulated in left ventricle outflow tract-derived MiVEC of AS ( P <0.05 versus right atrium-MiVEC). Fatty acid oxidation increased >2-fold in left ventricle outflow tract-MiVEC, proline content by 130% (median, IQR, 58%–474%; P =0.008) and procollagen secretion by 85% (mean [95% CI, 16%–154%]; P <0.05 versus right atrium-MiVEC for all). The altered transcriptome in left ventricle outflow tract-MiVEC was associated with impaired 2-dimensional-vascular network formation and 3-dimensional-spheroid sprouting ( P <0.05 versus right atrium-MiVEC), profibrotic ultrastructural changes, and impaired diastolic left ventricle function, capillary density and functional status, especially in female AS. Conclusions: Pressure overload induces major transcriptional and metabolic adaptations in cardiac MiVEC resulting in excess interstitial fibrosis and impaired angiogenesis. Molecular rewiring of MiVEC is worse in women, compromises functional status, and identifies novel targets for intervention.