Clinical and epidemiologic data in coronavirus disease (COVID-19) have accrued rapidly since the outbreak, but few address the underlying pathophysiology.
To ascertain the physiologic, hematologic, ...and imaging basis of lung injury in severe COVID-19 pneumonia.
Clinical, physiologic, and laboratory data were collated. Radiologic (computed tomography (CT) pulmonary angiography
= 39 and dual-energy CT DECT,
= 20) studies were evaluated: observers quantified CT patterns (including the extent of abnormal lung and the presence and extent of dilated peripheral vessels) and perfusion defects on DECT. Coagulation status was assessed using thromboelastography.
In 39 consecutive patients (male:female, 32:7; mean age, 53 ± 10 yr range, 29-79 yr; Black and minority ethnic,
= 25 64%), there was a significant vascular perfusion abnormality and increased physiologic dead space (dynamic compliance, 33.7 ± 14.7 ml/cm H
O; Murray lung injury score, 3.14 ± 0.53; mean ventilatory ratios, 2.6 ± 0.8) with evidence of hypercoagulability and fibrinolytic "shutdown". The mean CT extent (±SD) of normally aerated lung, ground-glass opacification, and dense parenchymal opacification were 23.5 ± 16.7%, 36.3 ± 24.7%, and 42.7 ± 27.1%, respectively. Dilated peripheral vessels were present in 21/33 (63.6%) patients with at least two assessable lobes (including 10/21 47.6% with no evidence of acute pulmonary emboli). Perfusion defects on DECT (assessable in 18/20 90%) were present in all patients (wedge-shaped,
= 3; mottled,
= 9; mixed pattern,
= 6).
Physiologic, hematologic, and imaging data show not only the presence of a hypercoagulable phenotype in severe COVID-19 pneumonia but also markedly impaired pulmonary perfusion likely caused by pulmonary angiopathy and thrombosis.
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.
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.
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.
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.
A number of adhesion-mediated signaling pathways and cell-cycle events have been identified that regulate cell proliferation, yet studies to date have been unable to determine which of these pathways ...control mitogenesis in response to physiologically relevant changes in tissue elasticity. In this report, we use hydrogel-based substrata matched to biological tissue stiffness to investigate the effects of matrix elasticity on the cell cycle.
We find that physiological tissue stiffness acts as a cell-cycle inhibitor in mammary epithelial cells and vascular smooth muscle cells; subcellular analysis in these cells, mouse embryonic fibroblasts, and osteoblasts shows that cell-cycle control by matrix stiffness is widely conserved. Remarkably, most mitogenic events previously documented as extracellular matrix (ECM)/integrin-dependent proceed normally when matrix stiffness is altered in the range that controls mitogenesis. These include ERK activity, immediate-early gene expression, and cdk inhibitor expression. In contrast, FAK-dependent Rac activation, Rac-dependent cyclin D1 gene induction, and cyclin D1-dependent Rb phosphorylation are strongly inhibited at physiological tissue stiffness and rescued when the matrix is stiffened in vitro. Importantly, the combined use of atomic force microscopy and fluorescence imaging in mice shows that comparable increases in tissue stiffness occur at sites of cell proliferation in vivo.
Matrix remodeling associated with pathogenesis is in itself a positive regulator of the cell cycle through a highly selective effect on integrin-dependent signaling to FAK, Rac, and cyclin D1.
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.
Display omitted
•Contractile and proliferative smooth muscle cell phenotypes emerge from a null state•Emergence from the null state is mediated by ECM stiffness, Rac, and Rho•Differential translocation of YAP and TAZ by Rac controls end state emergence
Reversible differentiation/de-differentiation of smooth muscle cells plays a critical role in vascular biology and disease. Talwar et al. show that these differentiated and de-differentiated phenotypes emerge from a null state that is regulated by ECM stiffness and bidirectional effects of Rac on YAP and TAZ transcriptional coregulators.
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 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 myosln light chain kinase. The expression of arterial fibrillar collagens, the integrity of arterial elastin, or markers of SMC differentiation were not affected by deletion of SMC FAK. Our results connect cell contractility and SMC FAK to the regulation of arterial wall thickness and directionally-specific arterial stiffening.
Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple is unknown. Here, we show that apolipoprotein E (apoE) and apoE-containing high-density lipoprotein ...(apoE-HDL) maintain arterial elasticity by suppressing the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop that increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain and transduced by Cox2 and miR-145. Arterial stiffness is increased in apoE null mice. This stiffening can be reduced by administration of the lysyl oxidase inhibitor BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. We conclude that apoE and apoE-containing HDL promote healthy arterial biomechanics and that this confers protection from cardiovascular disease independent of the established apoE-HDL effect on cholesterol.
Display omitted
► ApoE-HDL inhibits ECM expression and arterial stiffening ► Substratum stiffening induces expression of collagen-I, fibronectin, and lysyl oxidase ► The suppressive effect of apoE on arterial stiffness is transduced by Cox2 and miR-145
Assoian and colleagues describe links between cardiovascular protection, HDL, the extracellular matrix, and arterial mechanics. They demonstrate that apoE and the subspecies of HDL containing apoE suppress collagen-I, fibronectin, and lysyl oxidase gene expression in vascular smooth muscle cells and in vivo. The effect is transduced by Cox2 and miR-145 and independent of the apoE lipid binding domain. Suppression of ECM gene expression by apoE-HDL limits arterial stiffening and protects against atherosclerosis even in the presence of high cholesterol.
In contrast to the accepted pro-proliferative effect of cell-matrix adhesion, the proliferative effect of cadherin-mediated cell-cell adhesion remains unresolved. Here, we studied the effect of ...N-cadherin on cell proliferation in the vasculature. We show that N-cadherin is induced in smooth muscle cells (SMCs) in response to vascular injury, an in vivo model of tissue stiffening and proliferation. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated activation of a focal adhesion kinase (FAK)-p130Cas-Rac signaling pathway induces N-cadherin. Additionally, by culturing paired and unpaired SMCs on microfabricated adhesive islands of different areas, we found that N-cadherin relaxes the spreading requirement for SMC proliferation. In vivo SMC deletion of N-cadherin strongly reduced injury-induced cycling. Finally, SMC-specific deletion of FAK inhibited proliferation after vascular injury, and this was accompanied by reduced induction of N-cadherin. Thus, a stiffness- and FAK-dependent induction of N-cadherin connects cell-matrix to cell-cell adhesion and regulates the degree of cell spreading needed for cycling.
Display omitted
•N-cadherin expression is regulated by stiffness-sensitive FAK signaling•N-cadherin is essential for in vivo vascular smooth muscle cell proliferation•N-cadherin overrides the spreading requirement for cell cycling
Mui et al. show that N-cadherin is induced by extracellular matrix stiffness and FAK. N-cadherin is essential for smooth muscle cell proliferation in vivo and when cell spreading is constrained in vitro. Their study reveals crosstalk between cell-cell and cell-substrate adhesions, as well as a basis for cadherin-mediated stimulation of proliferation.
Hyaluronan, a widely distributed component of the extracellular matrix, exists in a high molecular weight (native) form and lower molecular weight form (HMW- and LMW-HA, respectively). These ...different forms of hyaluronan bind to CD44 but elicit distinct effects on cellular function. A striking example is the opposing effects of HMW- and LMW-HA on the proliferation of vascular smooth muscle cells; the binding of HMW-HA to CD44 inhibits cell cycle progression, whereas the binding of LMW-HA to CD44 stimulates cell cycle progression. We now report that cyclin D1 is the primary target of LMW-HA in human vascular smooth muscle cells, as it is for HMW-HA, and that the opposing cell cycle effects of these CD44 ligands result from differential regulation of signaling pathways to cyclin D1. HMW-HA binding to CD44 selectively inhibits the GTP loading of Rac and Rac-dependent signaling to the cyclin D1 gene, whereas LMW-HA binding to CD44 selectively stimulates ERK activation and ERK-dependent cyclin D1 gene expression. These data describe a novel mechanism of growth control in which a ligand-receptor system generates opposing effects on mitogenesis by differentially regulating signaling pathways to a common cell cycle target. They also emphasize how a seemingly subtle change in matrix composition can have a profound effect on cell proliferation.
PDF
