Arterial stiffening is influenced by the organization of focal adhesions in vascular smooth muscle cells (VSMCs). We investigated the contribution of αv integrins to both arterial wall stiffness (Young’s modulus measured by echography) and VSMC stiffness (assessed by atomic force microscopy). Mice with VSMC-specific deletion of αv integrins (αvSMKO) were compared with controls at baseline and following angiotensin II infusion. Unstimulated cultured αv-deficient (αv-KD) VSMCs exhibited higher stiffness than controls, with a further increase after angiotensin II. To interpret AFM measurements performed at shallow indentation depths, we developed a computational model of VSMC nanoindentation. Simulations showed that higher apparent Young’s moduli at shallow indentation fall within the experimental range of αv-KD cells. These cells also displayed enhanced actin polymerization, further amplified by angiotensin II through the formation of cortical F-actin. In vivo, arterial pressure and wall elastic modulus were similar between αvSMKO and control mice at baseline and after angiotensin II, despite αvSMKO mice exhibiting lower elastin and higher collagen content under angiotensin II. Together, these findings indicate that the comparable increase in arterial stiffness observed in αvSMKO mice under angiotensin II is driven primarily by elevated VSMC stiffness resulting from cortical actin redistribution, which outweighs extracellular matrix changes.

Background:Human aging is characterized by endothelial dysfunction that drives a systemic prothrombotic shift. In contrast, the long-lived naked mole rat (NMR) represents a unique model of delayed aging, exhibiting a notable resistance to age-related pathologies. However, while its cardiovascular stability is well-documented, the NMR hemostatic profile across its lifespan remains unexplored. Objectives:To assess whether NMRs undergo age-related hypercoagulability and to compare their hemostatic trajectory with that of humans. Methods:We compared young (2-year-old) and aged (20-year-old) NMRs. Assessments included clotting factor quantification, endothelial markers, and integrative thrombin generation assays. Plasma from human volunteers (20-year-old vs 80-year-old NMRs) were used as a reference point for typical hemostatic aging. Results:NMRs maintained cellular blood composition and showed no age-related increase in markers of endothelial activation (including von Willebrand factor, factor VIII, tissue factor pathway inhibitor, soluble thrombomodulin, and tissue plasminogen activator). While aged NMRs showed a modest increase in fibrinogen and D-dimer, this rise was significantly lower than the 2- to 5-fold elevations seen in elderly humans. Most notably, thrombin generation potential remained identical between young and aged NMRs. In contrast, humans exhibited a marked age-dependent shift toward accelerated and heightened thrombin production. Conclusion:NMRs possess the ability to bypass the pathologic clotting shifts that drive thrombotic events in humans, effectively decoupling chronologic aging from prothrombotic risk. By maintaining stable endothelial coagulation markers and an unchanged thrombin-forming capacity throughout their lifespan, NMRs appear naturally protected against age-dependent hypercoagulability.

Vascular aging is considered now to be the first factor of multiorgan aging in what is called ‘the vascular theory of aging’. Clinical understanding of vascular aging has long been limited to arterial hypertension and arterial stiffness. The effects of age on arterial mechanical properties have always been difficult to interpret for reasons linked to the non-linear behaviour of the stiffness/pressure function and the complex interactions between vascular cells and the matrix. Even new methodologies for decoding aging at the single-cell level are equally difficult to interpret. The objectives of this review are: (i) to introduce new computational approaches in biomechanics and mechanobiology; (ii) to revisit the role of oxidative stress and cellular senescence; (iii) to summarize some of the main molecular, cellular, and mechanistic contributions to vascular aging; (iv) to present the latest human studies of accelerated arterial aging with particular reference to cognitive impairment and functional decline; and (v) to propose some future directions for research related to vascular aging.

Rationale: Pulmonary hypertension (PH) is characterized by extensive remodeling of small pulmonary vessels, in part driven by the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). Von Willebrand factor (vWF), a multifunctional glycoprotein, is elevated in PH patients and implicated in inflammation and thrombosis. However, its role in modulating PASMC proliferation in PH remains unclear. This study aims to elucidate the molecular mechanisms underlying vWF-induced PA-SMC proliferation in an in vitro model of PH. Methods: Human PA-SMCs were exposed to hypoxic conditions to replicate the PH microenvironment. The effects of vWF on cell proliferation and phenotypic markers were evaluated using proliferation assays (BrdU incorporation and cell counting), immunoblotting, and cell-based ELISA. To elucidate the underlying signaling pathways, cells were treated with αv integrin siRNA or an RGD-peptide inhibitor to specifically target αvβ3 integrin function. Additional experiments investigated the potential involvement of co-receptors and downstream signaling effectors. Results: After 48 hours of hypoxia, platelet-derived growth factor (PDGF) treatment resulted in a 1.36-fold increase in PA-SMC count. Notably, vWF treatment led to a more pronounced 1.73-fold increase. Statistical analysis revealed significant differences, with p < 0.0001 for VWF compared to the untreated control and p = 0.002 for vWF compared to PDGF. Concurrently, confocal microscopy confirmed the expression of αvβ3 and LRP4 on PA-SMCs, suggesting a potential colocalization that may activate proliferative signaling pathways.Inhibition of αvβ3 integrin significantly attenuated vWF-induced PA-SMC proliferation. Silencing αvβ3 with siRNA reduced the proliferation index of vWF-treated PA-SMCs from 2.35-fold to 1.45-fold compared to control siRNA-treated cells (p = 0.0013) (Figure 1). No significant difference was observed between groups treated with αv siRNA, regardless of vWF treatment, highlighting the essential role of αvβ3 in vWF-mediated proliferation. Similar results were observed with the RGD-peptide inhibitor.Phosphorylation of Src, ERK, and Akt was assessed at various time points post-vWF treatment. Inhibition of αvβ3 led to reduced phosphorylation of these signaling proteins, suggesting that vWF activates these pathways through an αvβ3-dependent mechanism. Furthermore, overexpression of osteopontin was observed in vWF-treated PA-SMCs under chronic hypoxia, indicating its involvement in vascular remodeling. Conclusions: Our findings identify a novel role for vWF in promoting PA-SMC proliferation via αvβ3 integrin signaling. The identification of the vWF/LRP/αvβ3 axis provides a potential therapeutic target for PH. Targeting vWF or αvβ3 integrin could represent a promising strategy to mitigate vascular remodeling and disease progression in PH.

Macromolecular crowding (MMC) is a ubiquitous phenomenon in biological systems that is largely overlooked in bioengineered in vitro cellular models. This comprehensive review examines the significant impact of both intracellular and extracellular MMC on cellular and molecular processes under physiological and pathological conditions. By synthesizing current knowledge and identifying critical gaps in our understanding of MMC, this review highlights the need to incorporate crowding into the development of in vitro models for studying health and diseases, as well as for drug discovery platforms. The pervasive nature of MMC in biological systems underscores its potential importance in various physiological and pathological processes, including protein aggregation disorders, cancer, and vascular diseases. Recognizing the ubiquitous influence of MMC could open new avenues for therapeutic interventions and deepen our understanding of fundamental biological processes.

Aneurysms of the thoracic (TAA) and abdominal aorta (AAA) have different pathophysiological mechanisms. AAA has an intraluminal thrombus, while TAA does not. This suggests a prothrombotic phenotype in AAA, probably at the level of vascular smooth muscle cells (SMCs) known to express tissue factor (TF).To explore the TF-dependent thrombin generation in SMCs in AAA compared with TAA and healthy aorta (HA) and the underlying mechanisms contributing to a procoagulant phenotype.Human HA, AAA, or TAA tissues and corresponding SMC primary cultures were used to analyze SMC-supported thrombin generation and TF expression.In the absence of added TF, thrombin generation was increased at the surface of SMCs from AAA compared with TAA and HA, indicating a cellular procoagulant phenotype, which is transmitted through mitosis. Phosphatidylserine exposure was increased at the surface of SMCs from AAA. As expected, reactive oxygen species generation and the proinflammatory cytokine TNF-α were increased in SMCs from AAA. Overexpression of protease-activated receptor 2 and nuclear translocation of NF-κB p65 in SMCs and tissue from AAA triggered increased TF gene expression. Higher active basal TF expression was also observed in SMCs from AAA, which was inhibited by BAY 11-7082 (pharmacological inhibitor of IκK) and GB83 (pharmacological inhibitor of PAR-2).We demonstrated a PAR-2-mediated activation of the canonical NF-κB pathway, which triggers TF transcription in AAA. This procoagulant profile is transmitted from tissue to primary SMC cultures and through numerous passages, which can maintain thrombus formation.

Les propriétés élastiques des artères de conductance constituent l’une des fonctions hémodynamiques les plus importantes de l’organisme. Des données nouvelles continuent d’émerger sur l’importance de leurs dysfonctionnements dans le vieillissement vasculaire et la plupart des maladies cardiovasculaires communes. Nous examinerons les progrès réalisés sur les mécanismes biologiques qui apparaissent aujourd’hui comme d’importants déterminants fondamentaux de la rigidité artérielle, en particulier ceux qui interviennent dans la contractilité, la plasticité et la rigidité des cellules musculaires lisses vasculaires (CMLVs). Nous nous concentrerons sur la mécanotransduction membranaire et nucléaire, la fonction de phagocytose et les stimuli immuno-inflammatoires. Enfin, nous discuterons des avancées au plan méthodologique, diagnostique et thérapeutique en insistant sur les questions de co-morbidité et la prise en charge individuelle des sujets.

ObjectivesThe main challenge in the care of patients with primary antiphospholipid syndrome (APS) or associated to systemic lupus erythematosus (SLE) is to determine whether patients will experience new events that may impair their clinical outcome. Triggering receptor expressed on myeloid cells-1 (TREM-1) is an amplifier of the Toll like receptor (TLR4) pathway, which is involved in APS. Plasma soluble TREM-1 (sTREM-1) levels indicate increased receptor activation and were significantly greater in thrombotic primary APS patients compared to controls. This prospective cohort study investigated the predictive value of plasma sTREM-1 levels at inclusion for the occurrence of thrombotic events or death in patients with APS, antiphospholipid antibodies (aPLs) and/or SLE.MethodsSerum sTREM-1 levels were measured at inclusion in 108 patients with APS, isolated aPL or SLE followed during 46 months. The primary outcomes included thrombosis, death and obstetrical morbidity. The occurrence of the first event of interest and predictors were modeled in a multivariable Cox model.ResultsDuring follow-up, 15 of the 108 patients presented with thromboses (14%), 5 patients died (5%), and 3 women experienced obstetrical morbidities (3%). Elevated serum sTREM-1 levels were an independent predictor for the occurrence of the composite outcome (HR 7.54 [95% CI; 2.44-23.31] p < .001). In addition, sTREM-1 levels were greater in patients with APS than patients with isolated aPL (p < .01).ConclusionHigh levels of sTREM-1 at inclusion predicted the occurrence of a thrombotic and obstetric event or death in patients with aPL and/or SLE. Therefore, sTREM-1 represents a potential new prognostic biomarker in these patients.

Ageing of the cardiovascular system is associated with frailty and various life-threatening diseases. As global populations grow older, age-related conditions increasingly determine healthspan and lifespan. The circulatory system not only supplies nutrients and oxygen to all tissues of the human body and removes by-products but also builds the largest interorgan communication network, thereby serving as a gatekeeper for healthy ageing. Therefore, elucidating organ-specific and cell-specific ageing mechanisms that compromise circulatory system functions could have the potential to prevent or ameliorate age-related cardiovascular diseases. In support of this concept, emerging evidence suggests that targeting the circulatory system might restore organ function. In this Roadmap, we delve into the organ-specific and cell-specific mechanisms that underlie ageing-related changes in the cardiovascular system. We raise unanswered questions regarding the optimal design of clinical trials, in which markers of biological ageing in humans could be assessed. We provide guidance for the development of gerotherapeutics, which will rely on the technological progress of the diagnostic toolbox to measure residual risk in elderly individuals. A major challenge in the quest to discover interventions that delay age-related conditions in humans is to identify molecular switches that can delay the onset of ageing changes. To overcome this roadblock, future clinical trials need to provide evidence that gerotherapeutics directly affect one or several hallmarks of ageing in such a manner as to delay, prevent, alleviate or treat age-associated dysfunction and diseases. In this Roadmap, Stellos and colleagues discuss the mechanisms of cardiovascular system ageing and how the ageing of blood, vessels and heart relates to the decline in organ function, and highlight potential therapeutic interventions, challenges in ageing research and future directions for preclinical and clinical studies.

Objective: Although the risk for thrombosis is well documented for inflammatory bowel disease (IBD) patients, the underlying pathological mechanism seems to be different from other thrombotic conditions. Deciphering the actors responsible for the increased risk of thrombosis in IBD would help to improve management of this frequent complication.Design: We studied the interplay between platelets, coagulation, and von Willebrand factor (VWF) in 193 IBD patients and in experimental models (acute and chronic) of colitis in wild-type and VWF-deficient mice.Results: We found a platelet-dependent increase in thrombin generation in IBD patients and in our mouse model of colitis. Agglutinated platelets were present in the blood of patients and mice. Interestingly, we observed not only a significant increase in total VWF antigen, but we were able to detect the presence of active VWF (VWF in its platelet-binding conformation; 3.2 +/- 2.7 mu g/ml) in the plasma of 30% of all IBD patients. In healthy controls, active VWF levels were below 0.3 mu g/ml. This led us to further explore experimental colitis in VWF-deficient mice and we observed that these mice were protected against the procoagulant state triggered by the colitis. Unexpectedly, these mice also manifested a significant worsening of colitis severity both in acute and chronic models.Conclusion: Platelets and VWF (including its active form) appear to be central players in the procoagulant phenotype in IBD. We observed that the role of VWF in hemostasis differs from its role in colic tissue healing, potentially opening new therapeutic avenues for a life-threatening complication in IBD patients.

Objective: Multiple mechanisms may contribute to a hypercoagulation state in patients with COVID-19. Endothelial cells play a key role in cellular adhesion, coagulation, smooth muscle cell proliferation, and vascular wall inflammation. We aimed to describe the activation of the coagulation system, specifically thrombin generation, plasma release of endothelial factors and NETosis, and their association with kidney function following the resolution of the acute phase of SARS-CoV-2 infection. Design and method: Plasma samples were collected from adults 3 to 9 months after a confirmed episode of COVID-19 (COVID+, hospitalized or not, n=22) or after negative testing for COVID-19 in individuals who remained free of COVID-19 (COVID-, n-22). The coagulation system was assessed using in-vivo markers such as D-dimers (ng/ml). Thrombin generation assay estimated the thrombin-generating capacity in platelet-rich and platelet-poor plasma to provide information on soluble coagulation factors and the role of platelets in this process. Plasma release of endothelial factors was assessed by the levels of procoagulant factor VIII (FVIII) and Von Willebrand factor (VWF), and NETosis was determined using DNA-histone complexes levels. Spearman and linear regressions were used, when appropriate, to assess relationships between coagulation biomarkers and clinical and demographic characteristics of the population, such as kidney function (eGFR) calculated with the CKD-EPI equation (ml/min/1.73m2). Results: The COVID+ group was older (mean age 53.8± 17.6 years vs 40.6 ± 17.2 years, p=0.031). Compared to the controls, the COVID+ group had higher DNA-histone levels (Spearman ρ =0.311, p=0.040) and lower platelet counts (Spearman ρ = -0.334, p=0.033), but these differences were no longer significant after adjustment for age. However, we observed a significant association between eGFR and D-dimers, VWF and FVIII. Among them, only the association of D-dimers with eGFR was independent of age (β=4.59, p=0.030). Conclusions: These findings do not support a persistent long-term dysregulation of coagulation and endothelial dysfunction following the resolution of COVID-19. However, they show a significant increase in D-dimers associated with renal function, independent of both age and COVID-19.

The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Platelet/leukocyte–endothelial interactions play a crucial role at the early stages of age-related cardiovascular diseases. Aging is associated with chronic inflammation promoting the adhesiveness of platelet and leukocytes at the vessel wall. Various platelet receptors and adhesive proteins drive these processes, which also depends on the rigidity of the vessel wall and on the local blood flow and shear conditions. Most of the inflammatory conditions enhance activation of platelets, leukocytes, or endothelial cells. Endothelial activation leads to release of von Willebrand factor and P-selectin, which can recruit and activate platelets and neutrophils. Activated platelets interact with innate and adaptive immune cells, leading to the formation of extracellular traps, in particular neutrophil extracellular traps, which in turn can trigger platelet activation. In addition, circulating cell-derived extracellular vesicles and platelet-derived miRs are becoming increasingly involved in inflammatory responses. The key pathways involved in platelet–leukocyte crosstalk and adhesion to endothelial cells are the basic mechanisms for developing more tailored therapies with minimized bleeding risk.

BACKGROUND:The effect of factor VIII (FVIII) or emicizumab on thrombin generation is usually assessed in assays using synthetic phospholipids. Here, we assessed thrombin generation at the surface of human arterial cells (aortic endothelial cells [hAECs] and aortic vascular smooth muscle cells [hVSMCs]). OBJECTIVES:To explore the capacity of hAECs (resting or stimulated) and hVSMCs to support thrombin generation by FVIII or emicizumab. METHODS:Primary hVSMCs and hAECs were analyzed for tissue factor (TF)-activity and antigen, phosphatidylserine (PS)-exposure, tissue factor pathway inhibitor (TFPI)-content and thrombomodulin expression. Cells were incubated with FVIII-deficient plasma spiked with FVIII, emicizumab, activated prothrombin complex concentrate (APCC) or combinations thereof. RESULTS:TF activity and PS-exposure were present on both hVSMCs and hAECs. In contrast, thrombomodulin and TFPI were expressed on hAECs, while virtually lacking on hVSMCs, confirming the procoagulant nature of hVSMCs. Tumor necrosis factor α-mediated stimulation of hAECs increased not only TF antigen, TF activity, and PS-exposure but also TFPI and thrombomodulin expression. As expected, FVIII and emicizumab promoted thrombin generation on nonstimulated hAECs and hVSMCs, with more thrombin being generated on hVSMCs. Unexpectedly, FVIII and emicizumab increased thrombin generation to a lesser extent on stimulated hAECs compared with nonstimulated hAECs. Finally, adding emicizumab to FVIII did not further increase thrombin generation, whereas the addition of emicizumab to APCC resulted in exaggerated thrombin generation. CONCLUSION:Tumor necrosis factor stimulation of hAECs increases both pro- and anticoagulant activity. Unexpectedly, the increased anticoagulant activity is sufficient to limit both FVIII- and emicizumab-induced thrombin generation. This protective effect disappears when emicizumab is combined with APCC.