Arterial mechanics, extracellular matrix, and smooth muscle differentiation in carotid arteries deficient for Rac1.

Stiffening of the extracellular matrix (ECM) occurs after vascular injury and contributes to the injury-associated proliferation of vascular smooth muscle cells (SMCs). ECM stiffness also activates Rac-GTP, and SMC Rac1 deletion strongly reduces the proliferative response to injury in vivo . However, ECM stiffening and Rac can affect SMC differentiation, which, in itself, can influence ECM stiffness and proliferation. Here, we used pressure myography and immunofluorescence analysis of mouse carotid arteries to ask if the reported effect of Rac1 deletion on in vivo SMC proliferation might be secondary to a Rac effect on basal arterial stiffness or SMC differentiation. The results show that Rac1 deletion does not affect the abundance of arterial collagen-I, -III, or -V, the integrity of arterial elastin, or the arterial responses to pressure, including the axial and circumferential stretch-strain relationships that are assessments of arterial stiffness. Medial abundance of alpha-smooth muscle actin and smooth muscle-myosin heavy chain, markers of the SMC differentiated phenotype, were not statistically different in carotid arteries containing or deficient in Rac1. Nor did Rac1 deficiency have a statistically significant effect on carotid artery contraction to KCl. Overall, these data argue that the inhibitory effect of Rac1 deletion on in vivo SMC proliferation reflects a primary effect of Rac1 signaling to the cell cycle rather than a secondary effect associated with altered SMC differentiation or arterial stiffness.

Endothelial lipid droplets suppress eNOS to link high fat consumption to blood pressure elevation.

Metabolic syndrome, today affecting more than 20% of the US population, is a group of 5 conditions that often coexist and that strongly predispose to cardiovascular disease. How these conditions are linked mechanistically remains unclear, especially two of these: obesity and elevated blood pressure. Here, we show that high fat consumption in mice leads to the accumulation of lipid droplets in endothelial cells throughout the organism and that lipid droplet accumulation in endothelium suppresses endothelial nitric oxide synthase (eNOS), reduces NO production, elevates blood pressure, and accelerates atherosclerosis. Mechanistically, the accumulation of lipid droplets destabilizes eNOS mRNA and activates an endothelial inflammatory signaling cascade that suppresses eNOS and NO production. Pharmacological prevention of lipid droplet formation reverses the suppression of NO production in cell culture and in vivo and blunts blood pressure elevation in response to a high-fat diet. These results highlight lipid droplets as a critical and unappreciated component of endothelial cell biology, explain how lipids increase blood pressure acutely, and provide a mechanistic account for the epidemiological link between obesity and elevated blood pressure.

Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3.

The Rho family GTPases Rac and Rho play critical roles in transmitting mechanical information contained within the extracellular matrix (ECM) to the cell. Rac and Rho have well-described roles in regulating stiffness-dependent actin remodeling, proliferation and motility. However, much less is known about the relative roles of these GTPases in stiffness-dependent transcription, particularly at the genome-wide level. Here, we selectively inhibited Rac and Rho in mouse embryonic fibroblasts cultured on deformable substrata and used RNA sequencing to elucidate and compare the contribution of these GTPases to the early transcriptional response to ECM stiffness. Surprisingly, we found that the stiffness-dependent activation of Rac was dominant over Rho in the initial transcriptional response to ECM stiffness. We also identified activating transcription factor 3 (ATF3) as a major target of stiffness- and Rac-mediated signaling and show that ATF3 repression by ECM stiffness helps to explain how the stiffness-dependent activation of Rac results in the induction of cyclin D1.

Cell contractility and focal adhesion kinase control circumferential arterial stiffness.

Arterial stiffening is a hallmark of aging and cardiovascular disease. While it is well established that vascular smooth muscle cells (SMCs) contribute to arterial stiffness by synthesizing and remodeling the arterial extracellular matrix, the direct contributions of SMC contractility and mechanosensors to arterial stiffness, and particularly the arterial response to pressure, remain less well understood despite being a long-standing question of biomedical importance. Here, we have examined this issue by combining the use of pressure myography of intact carotid arteries, pharmacologic inhibition of contractility, and genetic deletion of SMC focal adhesion kinase (FAK). Biaxial inflation-extension tests performed at physiological pressures showed that acute inhibition of cell contractility with blebbistatin or EGTA altered vessel geometry and preferentially reduced circumferential, as opposed to axial, arterial stiffness in wild-type mice. Similarly, genetic deletion of SMC FAK, which attenuated arterial contraction to KCl, reduced vessel wall thickness and circumferential arterial stiffness in response to pressure while having minimal effect on axial mechanics. Moreover, these effects of FAK deletion were lost by treating arteries with blebbistatin or by inhibiting myosin light-chain kinase. The expression of arterial fibrillar collagens, the integrity of arterial elastin, or markers of SMC differentiation were not affected by the deletion of SMC FAK. Our results connect cell contractility and SMC FAK to the regulation of arterial wall thickness and directionally specific arterial stiffening.

Extra-cardiac BCAA catabolism lowers blood pressure and protects from heart failure.

Pharmacologic activation of branched-chain amino acid (BCAA) catabolism is protective in models of heart failure (HF). How protection occurs remains unclear, although a causative block in cardiac BCAA oxidation is widely assumed. Here, we use in vivo isotope infusions to show that cardiac BCAA oxidation in fact increases, rather than decreases, in HF. Moreover, cardiac-specific activation of BCAA oxidation does not protect from HF even though systemic activation does. Lowering plasma and cardiac BCAAs also fails to confer significant protection, suggesting alternative mechanisms of protection. Surprisingly, activation of BCAA catabolism lowers blood pressure (BP), a known cardioprotective mechanism. BP lowering occurred independently of nitric oxide and reflected vascular resistance to adrenergic constriction. Mendelian randomization studies revealed that elevated plasma BCAAs portend higher BP in humans. Together, these data indicate that BCAA oxidation lowers vascular resistance, perhaps in part explaining cardioprotection in HF that is not mediated directly in cardiomyocytes.

Progerin mislocalizes myocardin-related transcription factor in Hutchinson-Guilford Progeria syndrome.

Hutchinson-Guilford Progeria syndrome (HGPS) is a rare genetic disease of premature aging and early death due to cardiovascular disease. The arteries of HGPS children and mice are pathologically stiff, and HGPS mice also display reduced arterial contractility. We recently showed that reduced contractility is an early event in HGPS and linked to an aberrantly low expression of smooth muscle myosin heavy chain (smMHC). Here, we have explored the basis for reduced smMHC abundance and asked whether it is a direct effect of progerin expression or a longer-term adaptive response. Myh11, the gene encoding for smMHC, is regulated by myocardin-related transcription factors (MRTFs), and we show that HGPS aortas have a reduced MRTF signature. Additionally, smooth muscle cells (SMCs) isolated from HGPS mice display reduced MRTF nuclear localization. Acute progerin expression in WT SMCs phenocopied both the decrease in MRTF nuclear localization and expression of Myh11 seen in HGPS. Interestingly, RNA-mediated depletion of MRTF-A in WT SMCs reproduced the preferential inhibitory effect of progerin on Myh11 mRNA relative to Acta2 mRNA. Our results show that progerin expression acutely disrupts MRTF localization to the nucleus and suggest that the consequent decrease in nuclear coactivator activity can help to explain the reduction in smMHC abundance and SMC contractility seen in HGPS.

Decreased vascular smooth muscle contractility in Hutchinson-Gilford Progeria Syndrome linked to defective smooth muscle myosin heavy chain expression.

Children with Hutchinson-Gilford Progeria Syndrome (HGPS) suffer from multiple cardiovascular pathologies due to the expression of progerin, a mutant form of the nuclear envelope protein Lamin A. Progerin expression has a dramatic effect on arterial smooth muscle cells (SMCs) and results in decreased viability and increased arterial stiffness. However, very little is known about how progerin affects SMC contractility. Here, we studied the LaminAG609G/G609G mouse model of HGPS and found reduced arterial contractility at an early age that correlates with a decrease in smooth muscle myosin heavy chain (SM-MHC) mRNA and protein expression. Traction force microscopy on isolated SMCs from these mice revealed reduced force generation compared to wild-type controls; this effect was phenocopied by depletion of SM-MHC in WT SMCs and overcome by ectopic expression of SM-MHC in HGPS SMCs. Arterial SM-MHC levels are also reduced with age in wild-type mice and humans, suggesting a common defect in arterial contractility in HGPS and normal aging.

Mechanosensitive smooth muscle cell phenotypic plasticity emerging from a null state and the balance between Rac and Rho.

Reversible differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in vascular biology and disease. Changes in VSMC differentiation correlate with stiffness of the arterial extracellular matrix (ECM), but causal relationships remain unclear. We show that VSMC plasticity is mechanosensitive and that both the de-differentiated and differentiated fates are promoted by the same ECM stiffness. Differential equations developed to model this behavior predicted that a null VSMC state generates the dual fates in response to ECM stiffness. Direct measurements of cellular forces, proliferation, and contractile gene expression validated these predictions and showed that fate outcome is mediated by Rac-Rho homeostasis. Rac, through distinct effects on YAP and TAZ, is required for both fates. Rho drives the contractile state alone, so its level of activity, relative to Rac, drives phenotypic choice. Our results show how the cellular response to a single ECM stiffness generates bi-stability and VSMC plasticity.

Arterial stiffness and cardiac dysfunction in Hutchinson-Gilford Progeria Syndrome corrected by inhibition of lysyl oxidase.

Arterial stiffening and cardiac dysfunction are hallmarks of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), but the molecular regulators remain unknown. Here, we show that the LaminAG609G mouse model of HGPS recapitulates the premature arterial stiffening and early diastolic dysfunction seen in human HGPS. Lysyl oxidase (LOX) is up-regulated in the arteries of these mice, and treatment with the LOX inhibitor, ß-aminopropionitrile, improves arterial mechanics and cardiac function. Genome-wide and mechanistic analysis revealed reduced expression of the LOX-regulator, miR-145, in HGPS arteries, and forced expression of miR-145 restores normal LOX gene expression in HGPS smooth muscle cells. LOX abundance is also increased in the carotid arteries of aged wild-type mice, but its spatial expression differs from HGPS and its up-regulation is independent of changes in miR-145 abundance. Our results show that miR-145 is selectively misregulated in HGPS and that the consequent up-regulation of LOX is causal for premature arterial stiffening and cardiac dysfunction.

Fibroblast Activation Protein Regulates Lesion Burden and the Fibroinflammatory Response in Apoe-Deficient Mice in a Sexually Dimorphic Manner.

Fibroblast activation protein (FAP) has been established as an inducible and mesenchymal cell-specific mediator of disease progression in cancer and fibrosis. Atherosclerosis is a fibroinflammatory disease, and FAP was previously reported to be up-regulated in human atherosclerotic plaques compared with normal vessel. We investigated the spatial and temporal distribution of Fap-expressing cells in a murine model of atherosclerosis and used a genetic approach to determine if and how Fap affected disease progression. Fap was found to be expressed predominantly on vascular smooth muscle cells in lesions of athero-prone Apoe-/- mice. Global deletion of Fap (Fap-/-) in Apoe-/- mice accelerated atherosclerotic disease progression in both males and females, with the effect observed earlier in males. Sex-specific effects on lesion morphology were observed. Relative levels of extracellular matrix, fibrotic, and inflammatory cell content were comparable in lesions in male mice regardless of Fap status. In contrast, lesions in Fap-/- female mice were characterized by a more fibrotic composition due to a reduction in inflammation, specifically a reduction in Mox macrophages. Combined, these data suggest that Fap restrains the progression of atherosclerosis and may contribute to the sexually dimorphic susceptibility to atherosclerosis by regulating the balance between inflammation (an indicator of vulnerability to plaque rupture) and fibrosis (an indicator of plaque stability).

Cellular sensing of micron-scale curvature: a frontier in understanding the microenvironment.

The vast majority of cell biological studies examine function and molecular mechanisms using cells on flat surfaces: glass, plastic and more recently elastomeric polymers. While these studies have provided a wealth of valuable insight, they fail to consider that most biologically occurring surfaces are curved, with a radius of curvature roughly corresponding to the length scale of cells themselves. Here, we review recent studies showing that cells detect and respond to these curvature cues by adjusting and re-orienting their cell bodies, actin fibres and nuclei as well as by changing their transcriptional programme. Modelling substratum curvature has the potential to provide fundamental new insight into cell behaviour and function in vivo.

Vascular smooth muscle cell-specific progerin expression in a mouse model of Hutchinson-Gilford progeria syndrome promotes arterial stiffness: Therapeutic effect of dietary nitrite.

Vascular stiffness is a major cause of cardiovascular disease during normal aging and in Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic disorder caused by ubiquitous progerin expression. This mutant form of lamin A causes premature aging associated with cardiovascular alterations that lead to death at an average age of 14.6 years. We investigated the mechanisms underlying vessel stiffness in LmnaG609G/G609G mice with ubiquitous progerin expression, and tested the effect of treatment with nitrites. We also bred LmnaLCS/LCS Tie2Cre+/tg and LmnaLCS/LCS SM22αCre+/tg mice, which express progerin specifically in endothelial cells (ECs) and in vascular smooth muscle cells (VSMCs), respectively, to determine the specific contribution of each cell type to vascular pathology. We found vessel stiffness and inward remodeling in arteries of LmnaG609G/G609G and LmnaLCS/LCS SM22αCre+/tg , but not in those from LmnaLCS/LCS Tie2Cre+/tg mice. Structural alterations in aortas of progeroid mice were associated with decreased smooth muscle tissue content, increased collagen deposition, and decreased transverse waving of elastin layers in the media. Functional studies identified collagen (unlike elastin and the cytoskeleton) as an underlying cause of aortic stiffness in progeroid mice. Consistent with this, we found increased deposition of collagens III, IV, V, and XII in the media of progeroid aortas. Vessel stiffness and inward remodeling in progeroid mice were prevented by adding sodium nitrite in drinking water. In conclusion, LmnaG609G/G609G arteries exhibit stiffness and inward remodeling, mainly due to progerin-induced damage to VSMCs, which causes increased deposition of medial collagen and a secondary alteration in elastin structure. Treatment with nitrites prevents vascular stiffness in progeria.

MMP12 Deletion Preferentially Attenuates Axial Stiffening of Aging Arteries.

Arterial stiffening is a hallmark of aging, but how aging affects the arterial response to pressure is still not completely understood, especially with regard to specific matrix metalloproteinases (MMPs). Here, we performed biaxial inflation-extension tests on C57BL/6 mice to study the effects of age and MMP12, a major arterial elastase, on arterial biomechanics. Aging from 2 to 24 months leads to both circumferential and axial stiffening with stretch, and these changes are associated with an increased wall thickness, a decreased inner radius-wall thickness ratio, and a decreased in vivo axial stretch. Analysis of in vivo stretch and stress-stretch curves with arteries from age- and sex-matched wild-type (WT) and MMP12-null arteries demonstrates that MMP12 deletion attenuates age-dependent arterial stiffening, mostly in the axial direction. MMP12 deletion also prevents the aging-associated decrease in the in vivo stretch and, in general, leads to an axial mechanics phenotype characteristic of much younger mice. Circumferential arterial mechanics were much less affected by deletion of MMP12. We conclude that the induction of MMP12 during aging preferentially promotes axial arterial stiffening.

Cardiovascular protection in females linked to estrogen-dependent inhibition of arterial stiffening and macrophage MMP12.

Arterial stiffening is a consequence of aging and a cholesterol-independent risk factor for cardiovascular disease (CVD). Arterial stiffening and CVD show a sex bias, with men more susceptible than premenopausal women. How arterial stiffness and sex interact at a molecular level to confer risk of CVD is not well understood. Here, we used the sexual dimorphism in LDLR-null mice to show that the protective effect of female sex on atherosclerosis is linked to reduced aortic stiffness and reduced expression of matrix metalloproteinase-12 (MMP12) by lesional macrophages. Deletion of MMP12 in LDLR-null mice attenuated the male sex bias for both arterial stiffness and atherosclerosis, and these effects occurred despite high serum cholesterol. Mechanistically, we found that oxidized LDL stimulates secretion of MMP12 in human as well as mouse macrophages. Estrogen antagonizes this effect by downregulating MMP12 expression. Our data support cholesterol-independent causal relationships between estrogen, oxidized LDL-induced secretion of macrophage MMP12, and arterial stiffness that protect against atherosclerosis in females and emphasize that reduced MMP12 functionality can confer atheroprotection to males.

Edges impose planar alignment in nematic monolayers by directing cell elongation and enhancing migration.

Boundaries play an important role in the emergence of nematic order in classical liquid crystal systems; we explore their importance in adhesive cells that form active nematics. In particular, we study how cells are affected by an edge, which in our experiments is a boundary between adhesive and non-adhesive domains on a planar surface. We find that such edges induce elongation and direct the migration of isolated fibroblasts. In confluent monolayers, these elongated cells co-align and migrate to form an active, two-dimensional nematic structure in which edges enforce planar alignment and provide local slip to streams of cells that move along them. On an adhesive square island of dimensions 1 mm × 1 mm, cells near the edges in confluent nematic monolayers have enhanced alignment and velocity. The corners of the adhesive island seed defects with signs that depend on the direction of the motion of the streams of cells that meet there. Distortions emerge with rotations of -π/2 to form a -1/4 defect for streams that move clockwise or counterclockwise, and +π/2 to form a +1/4 defect for converging streams. We explore how cells transmit alignment information to each other in the absence of an edge by studying cell pairs and find that while such pairs do co-align, this alignment is only transient and short lived. These results shed light on the importance of edges in imposing nematic order in confluent monolayers and how edges can be used as tools to pattern the long-range organization of cells for tissue engineering applications.

Gaussian Curvature Directs Stress Fiber Orientation and Cell Migration.

We show that substrates with nonzero Gaussian curvature influence the organization of stress fibers and direct the migration of cells. To study the role of Gaussian curvature, we developed a sphere-with-skirt surface in which a positive Gaussian curvature spherical cap is seamlessly surrounded by a negative Gaussian curvature draping skirt, both with principal radii similar to cell-length scales. We find significant reconfiguration of two subpopulations of stress fibers when fibroblasts are exposed to these curvatures. Apical stress fibers in cells on skirts align in the radial direction and avoid bending by forming chords across the concave gap, whereas basal stress fibers bend along the convex direction. Cell migration is also strongly influenced by the Gaussian curvature. Real-time imaging shows that cells migrating on skirts repolarize to establish a leading edge in the azimuthal direction. Thereafter, they migrate in that direction. This behavior is notably different from migration on planar surfaces, in which cells typically migrate in the same direction as the apical stress fiber orientation. Thus, this platform reveals that nonzero Gaussian curvature not only affects the positioning of cells and alignment of stress fiber subpopulations but also directs migration in a manner fundamentally distinct from that of migration on planar surfaces.

Mining the Stiffness-Sensitive Transcriptome in Human Vascular Smooth Muscle Cells Identifies Long Noncoding RNA Stiffness Regulators.

OBJECTIVE: Vascular extracellular matrix stiffening is a risk factor for aortic and coronary artery disease. How matrix stiffening regulates the transcriptome profile of human aortic and coronary vascular smooth muscle cells (VSMCs) is not well understood. Furthermore, the role of long noncoding RNAs (lncRNAs) in the cellular response to stiffening has never been explored. This study characterizes the stiffness-sensitive (SS) transcriptome of human aortic and coronary VSMCs and identifies potential key lncRNA regulators of stiffness-dependent VSMC functions. APPROACH AND RESULTS: Aortic and coronary VSMCs were cultured on hydrogel substrates mimicking physiological and pathological extracellular matrix stiffness. Total RNAseq was performed to compare the SS transcriptome profiles of aortic and coronary VSMCs. We identified 3098 genes (2842 protein coding and 157 lncRNA) that were stiffness sensitive in both aortic and coronary VSMCs (false discovery rate <1%). Hierarchical clustering revealed that aortic and coronary VSMCs grouped by stiffness rather than cell origin. Conservation analyses also revealed that SS genes were more conserved than stiffness-insensitive genes. These VSMC SS genes were less tissue-type specific and expressed in more tissues than stiffness-insensitive genes. Using unbiased systems analyses, we identified MALAT1 as an SS lncRNA that regulates stiffness-dependent VSMC proliferation and migration in vitro and in vivo. CONCLUSIONS: This study provides the transcriptomic landscape of human aortic and coronary VSMCs in response to extracellular matrix stiffness and identifies novel SS human lncRNAs. Our data suggest that the SS transcriptome is evolutionarily important to VSMCs function and that SS lncRNAs can act as regulators of stiffness-dependent phenotypes.

Stiffness-dependent motility and proliferation uncoupled by deletion of CD44.

Information in the microenvironment guides complex cellular decisions such as whether or not to proliferate and migrate. The effects of soluble extracellular signals on these cellular functions are fairly well understood, but relatively little is known about how the extracellular matrix (ECM), and particularly the mechanical information in the ECM, guides these cellular decisions. Here, we show that CD44, a major receptor for the glycosaminoglycan ECM component hyaluronan, coordinates the motility and proliferative responses to ECM stiffening. We analyzed these cellular responses on fibronectin-coated polyacrylamide hydrogels prepared at a physiologic range of ECM stiffness and found that stiffening of the ECM leads to both cell cycling and cell motility in serum-stimulated primary mouse dermal fibroblasts. Remarkably, deletion of CD44 impaired stiffness-stimulated motility of the primary cells without affecting other hallmark cellular responses to ECM stiffening including cell spread area, stress fiber formation, focal adhesion maturation, and intracellular stiffening. Even stiffness-mediated cell proliferation was unaffected by deletion of CD44. Our results reveal a novel effect of CD44, which is imposed downstream of ECM-mechanosensing and determines if cells couple or uncouple their proliferative and motility responses to ECM stiffness.

Curvature and Rho activation differentially control the alignment of cells and stress fibers.

In vivo, cells respond to a host of physical cues ranging from substrate stiffness to the organization of micro- and nanoscale fibrous networks. We show that macroscale substrates with radii of curvature from tens to hundreds of micrometers influence cell alignment. In a model system of fibroblasts, isolated cells aligned strongly in the axial direction on cylinders with radii similar to the cell length and more weakly on cylinders of much larger radius. Isolated vascular smooth muscle cells did not align as effectively as fibroblasts. However, both cell types aligned robustly in weak curvature fields when in confluent monolayers. We identified two distinct populations of stress fibers in both cell types: long, apical stress fibers that aligned axially and short, basal stress fibers that aligned circumferentially. Circumferential alignment of the basal stress fibers is in apparent disagreement with a long-standing hypothesis that energetic penalties for bending enforce axial alignment on cylinders. To explore this phenomenon, we manipulated stress fibers by activating Rho, a small guanosine triphosphatase that regulates stress fiber assembly. In response, apical stress fibers disassembled, whereas basal stress fibers thickened and aligned more strongly in the circumferential direction. By activating Rho in confluent monolayers of vascular smooth muscle cells, we recapitulated the circumferential alignment pattern of F-actin within these cells that is observed in cylindrical vessels in vivo. In agreement with recent theory, these results suggest that stress fiber bending penalties are overcome when stress fiber contractility is enhanced and motivate deeper study of the mechanics of these distinct stress fiber populations.

Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and N-cadherin balance in mural cell-endothelial cell-regulated barrier function.

The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that N-cadherin expression in mural cells plays a key role in barrier function, as CRISPR-mediated knockout of N-cadherin in the mural cells led to loss of barrier function, and overexpression of N-cadherin in CHO cells promoted barrier function. In summary, this bicellular model demonstrates the continuous and rapid modulation of adhesive interactions between endothelial and mural cells and its impact on vascular barrier function and highlights an in vitro platform to study the biology of perivascular-endothelial interactions.