Pulmonary Angiopathy in Severe COVID-19: Physiologic, Imaging, and Hematologic Observations.

Rationale: Clinical and epidemiologic data in coronavirus disease (COVID-19) have accrued rapidly since the outbreak, but few address the underlying pathophysiology.Objectives: To ascertain the physiologic, hematologic, and imaging basis of lung injury in severe COVID-19 pneumonia.Methods: Clinical, physiologic, and laboratory data were collated. Radiologic (computed tomography (CT) pulmonary angiography [n = 39] and dual-energy CT [DECT, n = 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.Measurements and Results: In 39 consecutive patients (male:female, 32:7; mean age, 53 ± 10 yr [range, 29-79 yr]; Black and minority ethnic, n = 25 [64%]), there was a significant vascular perfusion abnormality and increased physiologic dead space (dynamic compliance, 33.7 ± 14.7 ml/cm H2O; 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, n = 3; mottled, n = 9; mixed pattern, n = 6).Conclusions: 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.

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.

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.

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.

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.

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.

Veno-venous extracorporeal membrane oxygenation for the acute respiratory distress syndrome: a bridge too far?

Veno-Venous Extracorporeal Membrane Oxygenation (VV-ECMO) provides a bridge to recovery in patients with acute respiratory failure due to the acute respiratory distress syndrome (ARDS). Survival in ARDS has improved over 15 years, and VV-ECMO may rescue even the most severe of these patients. Predictors of survival on ICU are based upon the principles of reversibility of the inciting aetiology, and premorbid 'reserve' - an imprecise term encompassing comorbidities and frailty. ECMO can support failing organs for prolonged periods, thus sometimes masking trajectories of decline, or unmasking irretrievable intrinsic conditions at a later time point in the critical illness. Clinicians are confronted with new on-treatment dilemmas: how long should we continue this high level of care? Will the patient's limited respiratory reserve manage off ECMO? Or are we hastening their demise? How long is it justifiable to keep someone on ECMO, if the predicted survival off is ultimately poor, but they are in a stable state whilst supported? The palliative withdrawal from ECMO is unchartered territory that requires further study. We describe two representative cases and discuss the wide ethical issues surrounding the initiation and withdrawal of ECMO.

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.

Six Month Mortality in Patients with COVID-19 and Non-COVID-19 Viral Pneumonitis Managed with Veno-Venous Extracorporeal Membrane Oxygenation.

A significant proportion of patients with COVID-19 develop acute respiratory distress syndrome (ARDS) with high risk of death. The efficacy of veno-venous extracorporeal membrane oxygenation (VV-ECMO) for COVID-19 on longer-term outcomes, unlike in other viral pneumonias, is unknown. In this study, we aimed to compare the 6 month mortality of patients receiving VV-ECMO support for COVID-19 with a historical viral ARDS cohort. Fifty-three consecutive patients with COVID-19 ARDS admitted for VV-ECMO to the Royal Brompton Hospital between March 17, 2020 and May 30, 2020 were identified. Mortality, patient characteristics, complications, and ECMO parameters were then compared to a historical cohort of patients with non-COVID-19 viral pneumonia. At 6 months survival was significantly higher in the COVID-19 than in the non-COVID-19 viral pneumonia cohort (84.9% vs. 66.0%, p = 0.040). Patients with COVID-19 had an increased Murray score (3.50 vs. 3.25, p = 0.005), a decreased burden of organ dysfunction (sequential organ failure score score [8.76 vs. 10.42, p = 0.004]), an increased incidence of pulmonary embolism (69.8% vs. 24.5%, p < 0.001) and in those who survived to decannulation longer ECMO runs (19 vs. 11 days, p = 0.001). Our results suggest that survival in patients supported with EMCO for COVID-19 are at least as good as those treated for non-COVID-19 viral ARDS.

Cell-type- and cell-cycle-specific anti-mitogenesis by cicaprost.

Stents eluting anti-proliferative drugs limit restenosis, but drugs commonly used to date are relatively non-specific cytostatic agents which inhibit proliferation of intimal endothelial cells as well as medial smooth muscle cells and may thereby contribute to the clinical complications associated with angioplasty. In an effort to identify a more specific anti-proliferative agent, we compared the effects of rapamycin to those of cicaprost, a mimetic of the naturally occurring anti-mitogen, PGI(2). Rapamycin and cicaprost were both strongly anti-mitogenic in vascular smooth muscle cells (VSMCs). But unlike rapamycin, cicaprost did not inhibit mitogenesis in aortic endothelial cells even when used at concentrations >10-fold higher than its ED(50) for VSMCs. Similarly, both rapamycin and cicaprost have been reported to regulate levels of the cdk inhibitor, p27(kip1). But rapamycin remained anti-mitogenic in p27(kip1)-null VSMCs whereas the anti-mitogenic effect of cicaprost was completely dependent on p27(kip1). We conclude that stable PGI(2) mimetics may be highly specific inhibitors of p27(kip1)-dependent VSMC proliferation after vascular injury.

The mSin3A chromatin-modifying complex is essential for embryogenesis and T-cell development.

The corepressor mSin3A is the core component of a chromatin-modifying complex that is recruited by multiple gene-specific transcriptional repressors. In order to understand the role of mSin3A during development, we generated constitutive germ line as well as conditional msin3A deletions. msin3A deletion in the developing mouse embryo results in lethality at the postimplantation stage, demonstrating that it is an essential gene. Blastocysts derived from preimplantation msin3A null embryos and mouse embryo fibroblasts (MEFs) lacking msin3A display a significant reduction in cell division. msin3A null MEFs also show mislocalization of the heterochromatin protein, HP1alpha, without alterations in global histone acetylation. Heterozygous msin3A(+/-) mice with a systemic twofold decrease in mSin3A protein develop splenomegaly as well as kidney disease indicative of a disruption of lymphocyte homeostasis. Conditional deletion of msin3A from developing T cells results in reduced thymic cellularity and a fivefold decrease in the number of cytotoxic (CD8) T cells, while helper (CD4) T cells are unaffected. We show that CD8 development is dependent on mSin3A at a step downstream of T-cell receptor signaling and that loss of mSin3A specifically decreases survival of double-positive and CD8 T cells. Thus, msin3A is a pleiotropic gene which, in addition to its role in cell cycle progression, is required for the development and homeostasis of cells in the lymphoid lineage.

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.

Genomic analysis of Andamanese provides insights into ancient human migration into Asia and adaptation.

To shed light on the peopling of South Asia and the origins of the morphological adaptations found there, we analyzed whole-genome sequences from 10 Andamanese individuals and compared them with sequences for 60 individuals from mainland Indian populations with different ethnic histories and with publicly available data from other populations. We show that all Asian and Pacific populations share a single origin and expansion out of Africa, contradicting an earlier proposal of two independent waves of migration. We also show that populations from South and Southeast Asia harbor a small proportion of ancestry from an unknown extinct hominin, and this ancestry is absent from Europeans and East Asians. The footprints of adaptive selection in the genomes of the Andamanese show that the characteristic distinctive phenotypes of this population (including very short stature) do not reflect an ancient African origin but instead result from strong natural selection on genes related to human body size.

N-Cadherin Induction by ECM Stiffness and FAK Overrides the Spreading Requirement for Proliferation of Vascular Smooth Muscle Cells.

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.

The influence of peri-incisional triamcinolone acetonide injection on wound edge apposition.

Local administration of corticosteroids has been demonstrated to have both beneficial and detrimental effects on wound healing. The advantages of limiting localized edema must be weighed against corticosteroids' disadvantageous inhibition of the normal growth factor profile production that is essential for would healing. A single-center prospective, randomized, controlled, single-blind study of 57 patients undergoing hair restoration surgery (HRS) by one of three different surgeons revealed: 1) no dehiscence along the donor wound; 2) no statistically significant difference (p < 0.05) in wound edge apposition noted between patients receiving or not receiving intralesional corticosteroids at any of the four measured scalp regions and 3) a non-statistically significant trend emerged suggesting the benefit of corticosteroid at the temples (points of decreased donor closing tension) versus its potential hindrance along the mastoids (points of increased tension). These results suggest that peri-incisional triamcinolone acetonide (PITMC) does not have a statistically significant effect on donor wound edge apposition within 8-10 days of HRS. A subtle, though not statistically significant, trend emerged demonstrating the benefit of PITMC with respect to early phase donor wound edge apposition in areas of least donor closing tension and the hindrance of PITMC in regions of increased tension.

A FAK-Cas-Rac-lamellipodin signaling module transduces extracellular matrix stiffness into mechanosensitive cell cycling.

Tissue and extracellular matrix (ECM) stiffness is transduced into intracellular stiffness, signaling, and changes in cellular behavior. Integrins and several of their associated focal adhesion proteins have been implicated in sensing ECM stiffness. We investigated how an initial sensing event is translated into intracellular stiffness and a biologically interpretable signal. We found that a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas), and the guanosine triphosphatase Rac selectively transduced ECM stiffness into stable intracellular stiffness, increased the abundance of the cell cycle protein cyclin D1, and promoted S-phase entry. Rac-dependent intracellular stiffening involved its binding partner lamellipodin, a protein that transmits Rac signals to the cytoskeleton during cell migration. Our findings establish that mechanotransduction by a FAK-Cas-Rac-lamellipodin signaling module converts the external information encoded by ECM stiffness into stable intracellular stiffness and mechanosensitive cell cycling. Thus, lamellipodin is important not only in controlling cellular migration but also for regulating the cell cycle in response to mechanical signals.

Acute abdominal pain and radiological pneumoperitoneum - always an indication for laparotomy?

Pneumoperitoneum in the presence of acute abdominal pain is well recognised as an indication for laparotomy. We present a case of acute abdominal pain in the presence of an incidental pneumoperitoneum secondary to the rupture of pneumatosis intestinalis. We will discuss the importance of clinical context in the diagnosis and management of pneumoperitoneum and pneumatosis intestinalis.