Extracellular vimentin is an attachment factor that facilitates SARS-CoV-2 entry into human endothelial cells.

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography-tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.

Tryptophan Metabolites Target Transmembrane and Immunoglobulin Domain-Containing 1 Signaling to Augment Renal Tubular Injury.

Chronic kidney disease (CKD) is characterized by the accumulation of uremic toxins and renal tubular damage. Tryptophan-derived uremic toxins [indoxyl sulfate (IS) and kynurenine (Kyn)] are well-characterized tubulotoxins. Emerging evidence suggests that transmembrane and immunoglobulin domain-containing 1 (TMIGD1) protects tubular cells and promotes survival. However, the direct molecular mechanism(s) underlying how these two opposing pathways crosstalk remains unknown. We posited that IS and Kyn mediate tubular toxicity through TMIGD1 and the loss of TMIGD1 augments tubular injury. Results from the current study showed that IS and Kyn suppressed TMIGD1 transcription in tubular cells in a dose-dependent manner. The wild-type CCAAT enhancer-binding protein ß (C/EBPß) enhanced, whereas a dominant-negative C/EBPß suppressed, TMIGD1 promoter activity. IS down-regulated C/EBPß in primary human renal tubular cells. The adenine-induced CKD, unilateral ureteric obstruction, and deoxycorticosterone acetate salt unilateral nephrectomy models showed reduced TMIGD1 expression in the renal tubules, which correlated with C/EBPß expression. C/EBPß levels negatively correlated with the IS and Kyn levels. Inactivation of TMIGD1 in mice significantly lowered acetylated tubulin, decreased tubular cell proliferation, caused severe tubular damage, and worsened renal function. Thus, the current results demonstrate that TMIGD1 protects renal tubular cells from renal injury in different models of CKD and uncovers a novel mechanism of tubulotoxicity of tryptophan-based uremic toxins.

Characterization of long-term survival in Medicare patients undergoing arteriovenous hemodialysis access.

BACKGROUND: Patients undergoing arteriovenous (AV) access creation for hemodialysis often have significant comorbidities. Our goal was to quantify the long-term survival and associated risks factors for long-term mortality in these patients to aid in optimization of goals and expectations. METHODS: The Vascular Implant Surveillance and Interventional Outcomes Network Vascular Quality Initiative Medicare linked data was used to assess long-term survival in the HD registry. Demographics, comorbidities, and interventions were recorded. Because the majority of hemodialysis patients are provided Medicare, Medicare linkage was used to obtain survival data. Multivariable analysis was used to identify independent associations with mortality. RESULTS: There were 13,945 AV access patients analyzed including 10,872 (78%) AV fistulas and 3073 (22%) AV grafts. The median age was 67 years and 56% of patients were male. Approximately one-third had a prior AV access and 44.7% had prior tunneled dialysis catheters. Patients receiving an AV fistula, compared with AV grafts, were more often younger, male, White, obese, independently ambulatory, preoperatively living at home, and less often have a prior AV access and tunneled dialysis catheters (P < .05 for all). The 5-year mortality overall was 62.9% with 61.2% for AV fistulas and 68.8% for AV grafts (P < .001). On multivariable analysis for 5 year mortality, nonambulatory status (hazard ratio [HR], 1.67; 95% confidence interval [CI], 1.53-1.83; P < .001), lower extremity access (HR, 1.67; 95% CI, 1.35-2.05; P < .001), human immunodeficiency virus or acquired immunodeficiency syndrome (HR, 1.44; 95% CI, 1.13-1.82; P < .001), White race (HR, 1.43; 95% CI, 1.35-1.51; P < .001), congestive heart failure (HR, 1.33; 95% CI, 1.26-1.41; P < .001), chronic obstructive pulmonary disease (HR, 1.23; 95% CI, 1.15-1.31; P < .001), and AV graft placement (HR, 1.12; 95% CI, 1.02-1.23, P = .016) were most associated with poor survival. Factors associated with improved survival were never smoking (HR, .73; 95% CI, 0.67-0.79; P < .001), prior/quit smoking (HR, .78; 95% CI, 0.72-0.84; P < .001), preoperative home living (HR, .75; 95% CI, 0.68-0.83; P < .001), and hypertension (HR, .89; 95% CI, 0.8-0.99; P = .03). CONCLUSIONS: Long-term survival in Medicare patients undergoing AV access creation is poor with nearly two-thirds of patients having died at 5 years. There are many modifiable risk factors that may improve survival in these patients and give an opportunity for transplantation.

Recent evaluation by nephrologists is associated with decreased incidence of tunneled dialysis catheter being used at the time of first arteriovenous access creation.

OBJECTIVE: Late primary care provider (PCP) or nephrologist evaluation of patients with progressive kidney disease may be associated with increased morbidity and mortality. Among patients undergoing initial arteriovenous (AV) access creation, we aimed to study the relationship of recent PCP and nephrologist evaluations with perioperative morbidity and mortality. METHODS: We performed a retrospective review of patients from 2014 to 2022 who underwent initial AV access creation at an urban, safety-net hospital. Univariable and multivariable analyses identified associations of PCP and nephrologist evaluations <1 year and <3 months before surgery, respectively, with hemodialysis initiation via tunneled dialysis catheters (TDCs), 90-day readmission, and 90-day mortality. RESULTS: Among 558 patients receiving initial AV access, mean age was 59.7 ± 14 years, 59% were female gender, and 60.6% were Black race. Recent PCP and nephrology evaluations occurred in 386 (69%) and 362 (65%) patients, respectively. On multivariable analysis, unemployed and uninsured statuses were associated with decreased likelihood of PCP evaluation (unemployment: odds ratio [OR], 0.51; 95% confidence interval [CI], 0.34-0.77; uninsured status: OR, 0.05; 95% CI, 0.01-0.45) and nephrologist evaluation (unemployment: OR, 0.63; 95% CI, 0.43-0.91; uninsured status: OR, 0.22; 95% CI, 0.06-0.83) (all P < .05). Social support was associated with increased likelihood of PCP evaluation (OR, 1.81; 95% CI, 1.07-3.08) (all P < .05). Hemodialysis was initiated with TDCs in 304 patients (55%). Older age (OR, 0.98; 95% CI, 0.96-0.99), obesity (OR, 0.38; 95% CI, 0.25-0.58), and nephrologist evaluation (OR, 0.12; 95% CI, 0.08-0.19) were independently associated with decreased hemodialysis initiation with TDCs in patients receiving an initial AV access (all P < .05). Ninety-day readmission occurred in 270 cases (48%). Cirrhosis (OR, 2.5; 95% CI, 1.03-6.03; P = .04), coronary artery disease (OR, 2.31; 95% CI, 1.5-3.57), prosthetic AV access (OR, 1.84; 95% CI, 1.04-3.26), and impaired ambulation (OR, 1.75; 95% CI, 1.15-2.66) were independently associated with increased readmission (all P < .05). Older age (OR, 0.98; 95% CI, 0.97-0.99), prior TDC (OR, 0.65; 95% CI, 0.45-0.94), and unemployment (OR, 0.58; 95% CI, 0.39-0.86) were associated with decreased readmission (all P < .05). Ninety-day mortality occurred in 1.6% of patients. Neither PCP nor nephrologist evaluation was associated with readmission or mortality. CONCLUSIONS: Recent nephrology evaluation was associated with reduced hemodialysis initiation with TDCs among patients undergoing initial AV access creation. Unemployed and uninsured statuses posed barriers to accessing nephrology care.

Thrombosis risk prediction in lymphoma patients: A multi-institutional, retrospective model development and validation study.

Venous thromboembolism (VTE) poses a significant risk to cancer patients receiving systemic therapy. The generalizability of pan-cancer models to lymphomas is limited. Currently, there are no reliable risk prediction models for thrombosis in patients with lymphoma. Our objective was to create a risk assessment model (RAM) specifically for lymphomas. We performed a retrospective cohort study to develop Fine and Gray sub-distribution hazard model for VTE and pulmonary embolism (PE)/ lower extremity deep vein thrombosis (LE-DVT) respectively in adult lymphoma patients from the Veterans Affairs national healthcare system (VA). External validations were performed at the Harris Health System (HHS) and the MD Anderson Cancer Center (MDACC). Time-dependent c-statistic and calibration curves were used to assess discrimination and fit. There were 10,313 (VA), 854 (HHS), and 1858 (MDACC) patients in the derivation and validation cohorts with diverse baseline. At 6 months, the VTE incidence was 5.8% (VA), 8.2% (HHS), and 8.8% (MDACC), respectively. The corresponding estimates for PE/LE-DVT were 3.9% (VA), 4.5% (HHS), and 3.7% (MDACC), respectively. The variables in the final RAM included lymphoma histology, body mass index, therapy type, recent hospitalization, history of VTE, history of paralysis/immobilization, and time to treatment initiation. The RAM had c-statistics of 0.68 in the derivation and 0.69 and 0.72 in the two external validation cohorts. The two models achieved a clear differentiation in risk stratification in each cohort. Our findings suggest that easy-to-implement, clinical-based model could be used to predict personalized VTE risk for lymphoma patients.

Redox Dysregulation of Vascular Smooth Muscle Sirtuin-1 in Thoracic Aortic Aneurysm in Marfan Syndrome.

BACKGROUND: Thoracic aortic aneurysms (TAAs) are abnormal aortic dilatations and a major cardiovascular complication of Marfan syndrome. We previously demonstrated a critical role for vascular smooth muscle (VSM) SirT1 (sirtuin-1), a lysine deacetylase, against maladaptive aortic remodeling associated with chronic oxidative stress and aberrant activation of MMPs (matrix metalloproteinases). METHODS: In this study, we investigated whether redox dysregulation of SirT1 contributed to the pathogenesis of TAA using fibrillin-1 hypomorphic mice (Fbn1mgR/mgR), an established model of Marfan syndrome prone to aortic dissection/rupture. RESULTS: Oxidative stress markers 3-nitrotyrosine and 4-hydroxynonenal were significantly elevated in aortas of patients with Marfan syndrome. Moreover, reversible oxidative post-translational modifications (rOPTM) of protein cysteines, particularly S-glutathionylation, were dramatically increased in aortas of Fbn1mgR/mgR mice, before induction of severe oxidative stress markers. Fbn1mgR/mgR aortas and VSM cells exhibited an increase in rOPTM of SirT1, coinciding with the upregulation of acetylated proteins, an index of decreased SirT1 activity, and increased MMP2/9 activity. Mechanistically, we demonstrated that TGFß (transforming growth factor beta), which was increased in Fbn1mgR/mgR aortas, stimulated rOPTM of SirT1, decreasing its deacetylase activity in VSM cells. VSM cell-specific deletion of SirT1 in Fbn1mgR/mgR mice (SMKO-Fbn1mgR/mgR) caused a dramatic increase in aortic MMP2 expression and worsened TAA progression, leading to aortic rupture in 50% of SMKO-Fbn1mgR/mgR mice, compared with 25% of Fbn1mgR/mgR mice. rOPTM of SirT1, rOPTM-mediated inhibition of SirT1 activity, and increased MMP2/9 activity were all exacerbated by the deletion of Glrx (glutaredoxin-1), a specific deglutathionylation enzyme, while being corrected by overexpression of Glrx or of an oxidation-resistant SirT1 mutant in VSM cells. CONCLUSIONS: Our novel findings strongly suggest a causal role of S-glutathionylation of SirT1 in the pathogenesis of TAA. Prevention or reversal of SirT1 rOPTM may be a novel therapeutic strategy to prevent TAA and TAA dissection/ruptures in individuals with Marfan syndrome, for which, thus far, no targeted therapy has been developed.

Alleviating iatrogenic effects of paclitaxel via antiinflammatory treatment.

BACKGROUND: Paclitaxel (PTX) is touted as an essential medicine due to its extensive use as a chemotherapeutic agent for various cancers and an antiproliferative agent for endovascular applications. Emerging studies in cardio-oncology implicate various vascular complications of chemotherapeutic agents. METHODS: We evaluated the inflammatory response induced by the systemic administration of PTX. The investigation included RNAseq analysis of primary human endothelial cells (ECs) treated with PTX to identify transcriptional changes in pro-inflammatory mediators. Additionally, we used dexamethasone (DEX), a well-known antiinflammatory compound, to assess its effectiveness in counteracting these PTX-induced changes. Further, we studied the effects of PTX on monocyte chemoattractant protein-1 (MCP-1) levels in the media of ECs. The study also extended to in vivo analysis, where a group of mice was injected with PTX and subsequently harvested at different times to assess the immediate and delayed effects of PTX on inflammatory mediators in blood and aortic ECs. RESULTS: Our RNAseq analysis revealed that PTX treatment led to significant transcriptional perturbations in pro-inflammatory mediators such as MCP-1 and CD137 within primary human ECs. These changes were effectively abrogated when DEX was administered. In vitro experiments showed a marked increase in MCP-1 levels in EC media following PTX treatment, which returned to baseline upon treatment with DEX. In vivo, we observed a threefold increase in MCP-1 levels in blood and aortic ECs 12 h post-PTX administration. Similar trends were noted for CD137 and other downstream mediators like tissue factor, vascular cell adhesion molecule 1, and E-selectin in aortic ECs. CONCLUSION: Our findings illustrate that PTX exposure induces an upregulation of atherothrombotic mediators, which can be alleviated with concurrent administration of DEX. Considering these observations, further long-term investigations should focus on understanding the systemic implications associated with PTX-based therapies and explore the clinical relevance of DEX in mitigating such risks.

Early Kidney Transplantation or Conversion to Peritoneal Dialysis after First-Time Arteriovenous Access Creation.

BACKGROUND: After autogenous arteriovenous (AV) access creation for end-stage renal disease, a majority of patients will continue on hemodialysis (HD), a minority will receive definitive treatment with kidney transplantation, and a subset of patients will convert to peritoneal dialysis (PD). Our goal was to identify patient factors associated with early transition from HD to either kidney transplantation or PD. METHODS: This is a case-control study of all patients with first-time AV access creation in the Vascular Quality Initiative (2011-2022) who had long-term follow-up. Patients who remained on HD after AV access creation were the control group while patients who received early kidney transplant or who converted to PD were the 2 case groups. Relationship among demographics, comorbidities, neighborhood social disadvantage, and functional status as they relate to renal replacement therapy modality was assessed. RESULTS: There were 19,782 patients included; the average age was 62 ± 15 years and 57% were male. During the follow-up period of a median 306 (71-403) days, 1.3% underwent a kidney transplantation and 2.3% underwent conversion to PD. On univariable analysis, rates of kidney transplantation or conversion to PD varied with race (P < 0.001), insurance status (P < 0.001), area deprivation index (ADI) quintile (P < 0.001), and several medical comorbidities. On multivariable analysis, impaired ambulation, current smoking, Medicaid or Medicare insurance, Black race, heart failure, body mass index, and older age were associated with decreased transplantation rates. Conversion to PD was associated with ADI Q5, Q4, and Q3. Decreased conversion to PD was associated with impaired ambulation, Hispanic ethnicity, Black race, former smoking, medication-controlled diabetes, and older age. CONCLUSIONS: Decreased kidney transplantation was associated with Black race and noncommercial health insurance but not ADI quintile, suggesting disparities exist beyond community-level access to care. Early kidney transplantation conveyed a 3-year survival benefit compared with HD and PD, which had similar survival. Furthermore work is required to increase access to kidney transplantation and PD.

High Prevalence of Adverse Social Determinants of Health in Dialysis Access Creation Patients in a Safety-Net Setting.

BACKGROUND: Patients receiving dialysis access surgery are often exposed to adverse social determinants of health (SDH) that negatively impact their care. Our goal was to characterize these factors experienced by our arteriovenous dialysis access patients and identify differences in health outcomes based on their SDH. METHODS: We performed a retrospective cohort study of all patients who underwent dialysis access creation (2017-2021) and were screened for SDH at a clinical visit (using THRIVE survey) implemented at an urban, safety-net hospital institution within 1 year of access creation. Demographics, procedural details, early postoperative outcomes, survey responses, and referral to our hospital's preventive food pantry were recorded. Univariable analysis and multivariable analyses were performed to assess for associations with key health outcomes. RESULTS: There were 190 patients who responded to the survey within 1 year of their operation. At least 1 adverse SDH was identified in 42 (22%) patients. Normalized to number of respondents for each question, adverse SDH identified were difficulty obtaining transportation to medical appointments (18%), food insecurity (16%), difficulty affording utilities (13%), difficulty affording medication (12%), unemployed and seeking employment (9%), unstable housing (7%), difficulty caring for family/friends (6%), and desiring more education (5%). There were 71 (37%) patients who received food pantry referrals. Mean age was 60 years and 38% of patients were female and 64% were Black. More than half of patients (57%) had a tunneled dialysis catheter (TDC) at the time of access creation. Dialysis accesses created were brachiocephalic (39%), brachiobasilic (25%), radiocephalic fistulas (16%), and arteriovenous grafts (14%). Thirty-day emergency department (ED) visits, 30-day readmissions, and 90-day mortality occurred in 23%, 21%, and 2%, respectively. On univariable and multivariable analyses, any adverse SDH determined on survey and food pantry referral were not associated with preoperative dialysis through TDCs, receiving nonautogenous dialysis access, 30-day ED visits and readmissions, or 90-day mortality. CONCLUSION: Nearly a quarter of dialysis access surgery patients at a safety-net hospital experienced adverse SDH and more than one-third received a food pantry referral. Most common difficulties experienced include difficulty obtaining transportation to medical appointments, food insecurity, and difficulty paying for utilities and medication. Although there were no differences in postoperative outcomes, the high prevalence of these adverse SDH warrants prioritization of resources in this population to ensure healthy equity and further investigation into their effects on health outcomes.

Pharmacologic Manipulation of Late SV40 Factor Suppresses Wnt Signaling and Inhibits Growth of Allogeneic and Syngeneic Colon Cancer Xenografts.

Aberrant hyperactivation of Wnt signaling, driven by nuclear ß-catenin in the colonic epithelium, represents the seminal event in the initiation and progression of colorectal cancer (CRC). Despite its established role in CRC tumorigenesis, clinical translation of Wnt inhibitors remains unsuccessful. Late SV40 factor (LSF; encoded by TFCP2) is a transcription factor and a potent oncogene. The current study identified a chemotype, named factor quinolinone inhibitors (FQIs), that specifically inhibits LSF DNA-binding, partner protein-binding, and transactivation activities. The role of LSF and FQIs in CRC tumor growth was examined. Herein, the study showed that LSF and ß-catenin interacted in several CRC cell lines irrespective of their mutational profile, which was disrupted by FQI2-34. FQI2-34 suppressed Wnt activity in CRC cells in a dose-dependent manner. Leveraging both allogeneic and syngeneic xenograft models showed that FQI2-34 suppressed CRC tumor growth, significantly reduced nuclear ß-catenin, and down-regulated Wnt targets such as axis inhibition protein 2 (AXIN-2) and SRY-box transcription factor 9, in the xenograft cells. FQI2-34 suppressed the proliferation of xenograft cells. Adenocarcinomas from a series of stage IV CRC patients revealed a positive correlation between LSF expression and Wnt targets (AXIN-2 and SRY-box transcription factor 9) within the CRC cells. Collectively, this study uncovers the Wnt inhibitory and CRC growth-suppressive effects of these LSF inhibitors in CRC cells, revealing a novel target in CRC therapeutics.

Intravenous sodium thiosulphate for vascular calcification of hemodialysis patients-a systematic review and meta-analysis.

BACKGROUND: Vascular calcification (VC) is a common comorbidity among patients with chronic kidney disease (CKD), indicating major cardiovascular events. This study aimed to evaluate the effects and safety of intravenous sodium thiosulphate (STS) for VC in CKD patients. METHODS: Electronic databases were searched for clinical trials that provided data comparing outcomes among patients treated with and without STS. The PRISMA guidelines were followed. Efficacy was assessed using calcification scores and arterial stiffness. Safety was examined by analyzing adverse symptoms, electrolytes and bone mineral density (BMD). Random-effects models were performed. Meta-regression and sensitivity analysis were done. The risk of bias was assessed using the Cochrane tools. RESULTS: Among the 5601 publications, 6 studies involving 305 participants (mean age: 56 years, male: 56.6%) with all participants on maintenance hemodialysis met eligibility criteria. For efficacy, the progression in Agatston scores in the coronary arteries [107 patients, mean difference (MD): -241.27, 95% confidence interval (95% CI): -421.50 to -61.03] and iliac arteries (55 patients, MD: -382.00, 95% CI: -751.07 to -12.93) was lower in the STS treated group compared with controls. The increase in pulse wave velocity was lower in the STS group (104 patients, MD: -1.29 m/s, 95% CI: -2.24 to -0.34 m/s). No association was found between the change in calcification scores and STS regimen. For safety, gastrointestinal symptoms (e.g. nausea) and increased anion gap acidosis were noted. No reduction in BMD by STS was observed. CONCLUSIONS: Intravenous STS may attenuate the progression of VC and arterial stiffness in hemodialysis patients. Large and well-designed randomized controlled trials are warranted.

Venous thromboembolism risk in cancer patients receiving first-line immune checkpoint inhibitor versus chemotherapy.

It remains unclear if immune checkpoint inhibitor (ICI) therapy is associated with higher rate of venous thromboembolism (VTE) compared with cytotoxic chemotherapy (chemo) in patients with comparable cancer type, staging, and comorbidities. Using the national Veterans Affairs healthcare system database from 2016 to 2021, we performed a propensity score (PS)-weighted retrospective cohort study to compare the incidence of VTE in patients with selected stage III/IV cancer receiving first-line ICI versus chemo. The PS model utilized overlap weights to balance age, sex, race, treatment year, VTE history, paralysis/immobilization, prolonged hospitalization, cancer type, staging, time between diagnosis and treatment, and National Cancer Institute comorbidity index. Weighted Cox regressions with robust standard error were used to assess the hazard ratio (HR) and 95% confidence interval (CI). We found that among comparable advanced cancers, first-line ICI (n = 1823) and first-line chemo (n = 6345) had similar rates of VTE (8.49% for ICI and 8.36% for chemo at 6 months). The weighted HR was 1.06 (95% CI 0.88-1.26) for ICI versus chemo. In a subgroup analysis restricted to lung cancers, first-line ICI/chemo (n = 828), ICI monotherapy (n = 428), and chemo monotherapy (n = 4371) had similar rates of VTE (9.60% for ICI/chemo, 10.04% for ICI, and 8.91% for chemo at 6 months). The weighted HR was 1.05 (95% CI 0.77-1.42) for ICI versus chemo, and 1.08 (95% CI 0.83-1.42) for ICI/chemo versus chemo. In conclusion, ICI as a systemic therapy has a similarly elevated risk as cytotoxic chemo for VTE occurrence in cancer patients. This finding can inform future prospective studies exploring thromboprophylaxis strategies.

SARS-CoV-2 Disrupts Proximal Elements in the JAK-STAT Pathway.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

Deep-Learning-Driven Quantification of Interstitial Fibrosis in Digitized Kidney Biopsies.

Interstitial fibrosis and tubular atrophy (IFTA) on a renal biopsy are strong indicators of disease chronicity and prognosis. Techniques that are typically used for IFTA grading remain manual, leading to variability among pathologists. Accurate IFTA estimation using computational techniques can reduce this variability and provide quantitative assessment. Using trichrome-stained whole-slide images (WSIs) processed from human renal biopsies, we developed a deep-learning framework that captured finer pathologic structures at high resolution and overall context at the WSI level to predict IFTA grade. WSIs (n = 67) were obtained from The Ohio State University Wexner Medical Center. Five nephropathologists independently reviewed them and provided fibrosis scores that were converted to IFTA grades: ≤10% (none or minimal), 11% to 25% (mild), 26% to 50% (moderate), and >50% (severe). The model was developed by associating the WSIs with the IFTA grade determined by majority voting (reference estimate). Model performance was evaluated on WSIs (n = 28) obtained from the Kidney Precision Medicine Project. There was good agreement on the IFTA grading between the pathologists and the reference estimate (κ = 0.622 ± 0.071). The accuracy of the deep-learning model was 71.8% ± 5.3% on The Ohio State University Wexner Medical Center and 65.0% ± 4.2% on Kidney Precision Medicine Project data sets. Our approach to analyzing microscopic- and WSI-level changes in renal biopsies attempts to mimic the pathologist and provides a regional and contextual estimation of IFTA. Such methods can assist clinicopathologic diagnosis.

Transmembrane and Immunoglobulin Domain Containing 1, a Putative Tumor Suppressor, Induces G2/M Cell Cycle Checkpoint Arrest in Colon Cancer Cells.

Colorectal cancer (CRC) is a leading nonfamilial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis are not fully understood. This study demonstrates that cell adhesion molecule transmembrane and immunoglobulin domain containing 1 (TMIGD1) are highly expressed in mouse and human normal intestinal epithelial cells. TMIGD1 knockout mice were developed, and the loss of TMIGD1 in mice was shown to result in the development of adenomas in small intestine and colon. In addition, the loss of TMIGD1 significantly impaired intestinal epithelium brush border membrane, junctional polarity, and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation and cell migration, arrests cell cycle at the G2/M phase, and induces expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, TMIGD1 is shown to be progressively down-regulated in sporadic human CRC, and its downregulation correlates with poor overall survival. The findings herein identify TMIGD1 as a novel tumor suppressor gene and provide new insights into the pathogenesis of colorectal cancer and a novel potential therapeutic target.

Indoleamine 2,3-dioxygenase-1, a Novel Therapeutic Target for Post-Vascular Injury Thrombosis in CKD.

BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.

Haploinsufficiency of Casitas B-Lineage Lymphoma Augments the Progression of Colon Cancer in the Background of Adenomatous Polyposis Coli Inactivation.

Casitas B-lineage lymphoma (c-Cbl) is a recently identified ubiquitin ligase of nuclear ß-catenin and a suppressor of colorectal cancer (CRC) growth in cell culture and mouse tumor xenografts. We hypothesized that reduction in c-Cbl in colonic epithelium is likely to increase the levels of nuclear ß-catenin in the intestinal crypt, augmenting CRC tumorigenesis in an adenomatous polyposis coli (APCΔ14/+) mouse model. Haploinsufficient c-Cbl mice (APCΔ14/+ c-Cbl+/-) displayed a significant (threefold) increase in atypical hyperplasia and adenocarcinomas in the small and large intestines; however, no differences were noted in the adenoma frequency. In contrast to the APCΔ14/+ c-Cbl+/+ mice, APCΔ14/+ c-Cbl+/- crypts showed nuclear ß-catenin throughout the length of the crypts and up-regulation of Axin2, a canonical Wnt target gene, and SRY-box transcription factor 9, a marker of intestinal stem cells. In contrast, haploinsufficiency of c-Cbl+/- alone was insufficient to induce tumorigenesis regardless of an increase in the number of intestinal epithelial cells with nuclear ß-catenin and SRY-box transcription factor 9 in APC+/+ c-Cbl+/- mice. This study demonstrates that haploinsufficiency of c-Cbl results in Wnt hyperactivation in intestinal crypts and accelerates CRC progression to adenocarcinoma in the milieu of APCΔ14/+, a phenomenon not found with wild-type APC. While emphasizing the role of APC as a gatekeeper in CRC, this study also demonstrates that combined partial loss of c-Cbl and inactivation of APC significantly contribute to CRC tumorigenesis.

Metabolites in a mouse cancer model enhance venous thrombogenicity through the aryl hydrocarbon receptor-tissue factor axis.

Patients with malignancy are at 4- to 7-fold higher risk of venous thromboembolism (VTE), a potentially fatal, yet preventable complication. Although general mechanisms of thrombosis are enhanced in these patients, malignancy-specific triggers and their therapeutic implication remain poorly understood. Here we examined a colon cancer-specific VTE model and probed a set of metabolites with prothrombotic propensity in the inferior vena cava (IVC) ligation model. Athymic mice injected with human colon adenocarcinoma cells exhibited significantly higher IVC clot weights, a biological readout of venous thrombogenicity, compared with the control mice. Targeted metabolomics analysis of plasma of mice revealed an increase in the blood levels of kynurenine and indoxyl sulfate (tryptophan metabolites) in xenograft-bearing mice, which correlated positively with the increase in the IVC clot size. These metabolites are ligands of aryl hydrocarbon receptor (AHR) signaling. Accordingly, plasma from the xenograft-bearing mice activated the AHR pathway and augmented tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) levels in venous endothelial cells in an AHR-dependent manner. Consistent with these findings, the endothelium from the IVC of xenograft-bearing animals revealed nuclear AHR and upregulated TF and PAI-1 expression, telltale signs of an activated AHR-TF/PAI-1 axis. Importantly, pharmacological inhibition of AHR activity suppressed TF and PAI-1 expression in endothelial cells of the IVC and reduced clot weights in both kynurenine-injected and xenograft-bearing mice. Together, these data show dysregulated tryptophan metabolites in a mouse cancer model, and they reveal a novel link between these metabolites and the control of the AHR-TF/PAI-1 axis and VTE in cancer.

Platelet Dysfunction and Thrombosis in JAK2V617F-Mutated Primary Myelofibrotic Mice.

OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.

Temporal and tissue-specific activation of aryl hydrocarbon receptor in discrete mouse models of kidney disease.

Emerging evidence in animal models of chronic kidney disease (CKD) implicates Aryl Hydrocarbon Receptor (AHR) signaling as a mediator of uremic toxicity. However, details about its tissue-specific and time-dependent activation in response to various renal pathologies remain poorly defined. Here, a comprehensive analysis of AHR induction was conducted in response to discrete models of kidney diseases using a transgenic mouse line expressing the AHR responsive-promoter tethered to a ß-galactosidase reporter gene. Following validation using a canonical AHR ligand (a dioxin derivative), the transgenic mice were subjected to adenine-induced and ischemia/reperfusion-induced injury models representing CKD and acute kidney injury (AKI), respectively, in humans. Indoxyl sulfate was artificially increased in mice through the drinking water and by inhibiting its excretion into the urine. Adenine-fed mice showed a distinct and significant increase in ß-galactosidase in the proximal and distal renal tubules, cardiac myocytes, hepatocytes, and microvasculature in the cerebral cortex. The pattern of ß-galactosidase increase coincided with the changes in serum indoxyl sulfate levels. Machine-learning-based image quantification revealed positive correlations between indoxyl sulfate levels and ß-galactosidase expression in various tissues. This pattern of ß-galactosidase expression was recapitulated in the indoxyl sulfate-specific model. The ischemia/reperfusion injury model showed increase in ß-galactosidase in renal tubules that persisted despite reduction in serum indoxyl sulfate and blood urea nitrogen levels. Thus, our results demonstrate a relationship between AHR activation in various tissues of mice with CKD or AKI and the levels of indoxyl sulfate. This study demonstrates the use of a reporter gene mouse to probe tissue-specific manifestations of uremia in translationally relevant animal models and provide hypothesis-generating insights into the mechanism of uremic toxicity that warrant further investigation.