Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Sonic hedgehog signaling inhibition provides opportunities for targeted therapy by sulforaphane in regulating pancreatic cancer stem cell self-renewal.

Dysregulation of the sonic hedgehog (Shh) signaling pathway has been associated with cancer stem cells (CSC) and implicated in the initiation of pancreatic cancer. Pancreatic CSCs are rare tumor cells characterized by their ability to self-renew, and are responsible for tumor recurrence accompanied by resistance to current therapies. The lethality of these incurable, aggressive and invasive pancreatic tumors remains a daunting clinical challenge. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN), an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN induced apoptosis by inhibition of BCL-2 and activation of caspases. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. We propose that pancreatic cancer preventative effects of SFN may result from inhibition of the Shh pathway. Thus Sulforaphane potentially represents an inexpensive, safe and effective alternative for the management of pancreatic cancer

Regulation of Shh pathway by SFN in pancreatic cancer cell lines <i>in vitro</i>.

<p>(<b>A–B</b>), Inhibition of components of sonic hedgehog pathway. ASPC1 and PANC1 were treated with sulforaphane (0–20 µM) for 24 h. The expression of Gli1, Gli2 and Smo was measured by qRT-PCR and normalized to GAPDH expression. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. $, @, and & = significantly different from control, P < 0.05.</p

Characterization of human pancreatic CSCs from human primary tumors.

<p>(A–D), Expression of pancreatic stem cell markers. Flow cytometric analysis of Pancreatic CSCs expressing was performed using stem cell markers CD44 -PE, ESA-PerCP, CD133-APC, CD24-FITC and appropriate controls. (E–F), Expression of pancreatic epithelial markers and drug resistance genes. Flow cytometric analysis of Pancreatic CD44⁺ESA⁺CD133⁺CD24⁺ CSCs also expressed CK19-PE and ABCG2-PE respectively.</p

Effects of SFN on Gli translocation and transcription.

<p>(<b>A</b>), The nuclear translocation of Gli1 and Gli2, was measured by immunocytochemistry. Pancreatic CSCs were treated with sulforaphane (0–20 µM) for 24 h. Cells were then stained with anti-Gli and Gli2 antibody (green fluorescence), and DAPI (red fluorescence). Merged images are shown, which indicate yellow-orange staining of Gli 1 and Gli2 located in the nucleus due to co-localization of green and red fluorescence. (<b>B</b>), Inhibition of Gli transcription. Pancreatic CSCs were transduced with Gli-responsive GFP/firefly luciferase viral particles (pGreen Fire1-Gli with EF1, System Biosciences). After transduction, culture medium was replaced and CSCs were treated with sulforaphane (0–20 µM) for 24 h. Gli-responsive reporter activity was measured using a luciferase assay (Promega Corporation). Data represent mean ± SD. @, %, and * = significantly different from control, P < 0.05.</p

Regulation of Bcl-2 expression, caspase-3/7 activity, and apoptosis by SFN on Pancreatic CSCs.

<p>(<b>A</b>), Effects of SFN on BcL-2expression. q-RT-PCR was performed to examine the expression of BcL-2. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. @, % and $= significantly different from control, P < 0.05. (<b>B</b>), Effects of SFN on caspase-3/7 activity. Pancreatic CSCs treated with SFN (0–20 µM) for 24 h, and caspase-3/7 activity was measured as per manufacturer's instructions. Data represent mean ± SD. @,$ = significantly different from control, P < 0.05. (<b>C</b>), Pancreatic CSCs were treated with SFN (0–20 µM), and cell lysates were collected and Immunobloted for anti- BCL-2, cleaved Caspase 3 or β-actin antibody. (<b>D</b>), Effects of SFN on apoptosis. Pancreatic CSCs were treated with SFN (0–20 µM) for 48 h, and apoptosis was measured by PI staining using flow cytometry.</p

Regulation of Hh target genes involved in the maintenance of pluripotency in pancreatic cancer stem cells.

<p>(<b>A–B</b>), Effects of SFN on expression of Hh target genes in the pancreatic CSCs. Real time PCR (q-RT-PCR) was performed to examine the expression of Nanog and Oct4 and data were normalized with GAPDH. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. @ and % = significantly different from control, P < 0.05. (<b>C</b>), Pancreatic CSCs were treated with SFN (0–20 µM), and cell lysates were collected and Western blot analysis was performed using anti- Nanog, Oct4 or β-actin antibody. (<b>D–E</b>), Effects of SFN on expression of Hh target genes involved in cell proliferation in the pancreatic CSCs. Real time PCR (q-RT-PCR) was performed to examine the expression of PDGFRα and Cyclin D1, involved in the maintenance of proliferation was analyzed and normalized with GAPDH. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. @, %, and $ = significantly different from control, P < 0.05. (<b>F</b>), Pancreatic CSCs were treated with SFN (0–20 µM), and cell lysates were collected and Immunobloted for anti- PDGFRα, Cyclin D1 or β-actin antibody.</p

Expression of Shh pathway in pancreatic CSCs.

<p>(<b>A–C</b>), Relative expression of various components of Shh pathway was quantified in human pancreatic cancer stem cells (PanCSC), human pancreatic normal stem cells (HNPSC) and human pancreatic normal ductal epithelial cells (HPNE). The expression of Shh genes was quantified using quantitative reverse transcriptase polymerase chain reaction real-time assay (q-RT-PCR), and normalized to GAPDH expression. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. %, and $ = significantly different from control, P < 0.05. (<b>D</b>), Immunoblotting of Gli1/2, Smo and β-actin of human pancreatic cancer stem cells (PanCSC), human pancreatic normal stem cells (HNPSC) and human pancreatic normal ductal epithelial cells (HPNE).</p