Citations

Traditionally associated with neuronal structure and function, neurotrophins (NTs; e.g. brain derived neurotrophic factor (BDNF), neurotrophin‐3 (NT3 )) and their receptors (tropomyosin related kinase; Trk) have been found in the adult lung, pulmonary artery and airway; however, their roles remain unclear. Based on previous findings of pulmonary hypertension in NT receptor knockout mice, we hypothesized that NTs induce production of nitric oxide (NO) in pulmonary endothelial cells (EC), thereby modulating pulmonary vascular tone. In human pulmonary artery EC loaded with the NO sensitive fluorescent dye DAF‐2, 50 ƒÝM ATP and both BDNF and NT3 (1 and 10 nM) acutely (<5 min) and substantially in increased fluorescence levels. Addition of the NO scavenger PTIO reversed these changes. Specificity of DAF2 for NO was tested using the short‐acting NO donor MAHMA‐NONOate, and the nitrous oxide donor Sulfa‐NONOate. Furthermore, NT‐induced elevation in NO was significantly reduced by pre‐exposure to the tyrosine kinase inhibitor K252A (100 nM, 20 min), the NOS inhibitors L‐NMMA or L‐NAME, and the Ca2+ chelator BAPTA. Acute NT exposure also increased eNOS phosphorylation. These results indicate that NT¡¦s such as BDNF and NT3 can acutely modulate endothelial NO production, and may contribute to pulmonary vasodilation. Supported by NIH grants HL088029, (YSP), HL56470 (YSP), and Mayo Graduate School (LWM).

Regulation of intracellular Ca2+ in human airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca2+ release/reuptake and plasma membrane (PM) Ca2+ influx, including store‐operated Ca2+ entry (SOCE) following SR Ca2+ depletion. Studies in human ASM have shown that siRNA suppression of stromal interaction molecule 1 (STIM1) and Orai1 decrease SOCE; however, their exact role in regulating Ca2+ are still under investigation. In human ASM, STIM1 and STIM2 were found exclusively in SR membrane, and Orai1 only in PM (especially caveolae). GFP‐tagged STIM1 transfection showed intracellular/SR localization at baseline, with increased peri‐PM aggregation following store depletion with 10ƒÝM cyclopiazonic acid (CPA). Such aggregation was prevented by microtubule inhibition (nocodazole), but not actin inhibition (phalloidin). FRET analysis revealed co‐localization of STIM1 with TRPC3 and Orai1 in CPA‐stimulated cells. In fura‐2 loaded ASM cells, SOCE was significantly reduced by STIM1 and Orai1 siRNA (less so by STIM2 siRNA). However, basal Ca2+ and agonist (ACh, histamine)‐induced Ca2+ responses were substantially modulated by STIM2 suppression. These results indicate that in human ASM, STIM1 mediates SR‐PM interactions in regulating SOCE, while STIM2 modulates sub‐maximal agonist‐induced changes in Ca2+. Supported by NIH grant HL74309 (GCS), HL090595 (CMP) and HL088029 (YSP).

Plasma membrane Ca 2+ influx and sarcoplasmic reticulum (SR) Ca 2+ release are key components of intracellular Ca 2+ regulation in airway smooth muscle (ASM). We and others have previously found that ASM Ca 2+ regulation involves propagating Ca 2+ oscillations via these mechanisms. Ca 2+ oscillations in non‐excitable cells involve influx via arachidonic acid (AA) regulated channels (ARC); however, little is known about ARC in ASM. In human ASM cells loaded with fura2 (5 μM), we found that 10 μM AA triggered Ca 2+ oscillations. OAG, oleic acid and palmitic acid (10 μM) failed to elicit Ca 2+ responses. Pre‐incubation with LaCl 3 1 μM and 1mM; 5 min) inhibited AA‐induced oscillations. Inhibition of SR Ca 2+ release (ryanodine 10 μM and IP3 channel inhibitor Xestospongin C 10 μM; 30 min), decreased Ca 2+ oscillation frequency and amplitude. Inhibitors of receptor‐operated channels (SKF‐96365; 10 μM), lipoxygenase (zileuton; 10 μM), or cyclooxygenase (indomethacin; 1 μM), did not affect oscillations. Finally, siRNA for Caveolin‐1, STIM1 and Orai3 (25nM) reduced frequency and amplitude of AA‐induced oscillations. These results indicate a novel caveolar Ca 2+ influx mechanism in human ASM mediated via AA that interacts with SR to produce repetitive SR Ca 2+ release. Such AA‐mediated influx could potentially contribute to increased Ca 2+ in ASM during inflammation. Supported by NIH grants HL090595 (CMP) and HL088029 (YSP)

Enhanced airway contractility by inflammatory cytokines involves increased intracellular Ca 2+ levels in airway smooth muscle (ASM). In this study, we examined the role of the bidirectional plasma membrane Na + /Ca 2+ exchanger (NCX) in human ASM Ca 2+ regulation following exposure to the pro‐inflammatory cytokines tumor necrosis factor alpha (TNFα) or interleukin‐13 (IL‐13). Both cytokines significantly increased NCX1 expression, with these effects inhibited by dexamethasone (NFκB inhibitor) or PD98059 (MAPK inhibitor). In human ASM cells loaded with the Ca 2+ indicator fura‐2, or the intracellular Na + indicator SBFI, NCX‐mediated Ca 2+ influx (with simultaneous Na + efflux) was enhanced by both cytokines. NCX‐mediated Ca 2+ efflux (with simultaneous Na + influx) was also enhanced, but not to the same extent. The NCX inhibitor KBR7943 as well as NCX1 siRNA inhibited cytokine enhancement of both modes NCX and of Ca 2+ responses to bronchoconstrictor agonists. These data indicate that a) NCX normally operates in human ASM; b) cytokines increase NCX expression, leading to greater NCX‐mediated Ca 2+ influx, thus contributing to enhanced Ca 2+ in inflamed ASM. Supported by NIH grants HL74309 (GCS), HL090595‐S2 (VS) and HL090595 (CMP).

Senescent cells exacerbate COVID-19 Cellular senescence is a state elicited in response to stress signals and is associated with a damaging secretory phenotype. The number of senescent cells increases with advanced age and this in turn drives age-related diseases. Camell et al. show that senescent cells have an amplified inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (see the Perspective by Cox and Lord). This response is communicated to nonsenescent cells, suppressing viral defense mechanisms and increasing the expression of viral entry proteins. In old mice infected with a SARS-CoV-2–related virus, treatment with senolytics to reduce the senolytic cell burden reduced mortality and increased antiviral antibodies. Science , abe4832, this issue p. eabe4832 ; see also abi4474, p. 281

Abstract Purpose: We recently reported the development of a cell-free DNA (cfDNA) targeted methylation (TM)-based sequencing approach for a multi-cancer early detection (MCED) test that includes cancer signal origin prediction. Here, we evaluated the prognostic significance of cancer detection by the MCED test using longitudinal follow-up data. Experimental Design: As part of a Circulating Cell-free Genome Atlas (CCGA) substudy, plasma cfDNA samples were sequenced using a TM approach, and machine learning classifiers predicted cancer status and cancer signal origin. Overall survival (OS) of cancer participants in the first 3 years of follow-up was evaluated in relation to cancer detection by the MCED test and clinical characteristics. Results: Cancers not detected by the MCED test had significantly better OS (P &lt; 0.0001) than cancers detected, even after accounting for other covariates, including clinical stage and method of clinical diagnosis (i.e., standard-of-care screening or clinical presentation with signs/symptoms). Additionally, cancers not detected by the MCED test had better OS than was expected when data were adjusted for age, stage, and cancer type from the Surveillance, Epidemiology, and End Results (SEER) program. In cancers with current screening options, the MCED test also differentiated more aggressive cancers from less aggressive cancers (P &lt; 0.0001). Conclusions: Cancer detection by the MCED test was prognostic beyond clinical stage and method of diagnosis. Cancers not detected by the MCED test had better prognosis than cancers detected and SEER-based expected survival. Cancer detection and prognosis may be linked by the underlying biological factor of tumor fraction in cfDNA.

AbstractAlthough bone marrow‐derived mesenchymal stem cells (BMCs) have been widely used in spinal fusion procedures, adipose‐derived stem cells (ASCs) offer a number of advantages as an alternative clinical cell source. This study directly compares the efficacy of ASCs and BMCs from the same donor animals to achieve successful fusion when combined with a clinical‐grade bone graft substitute in a rat lumbar fusion model. ASCs and BMCs were isolated from the same Lewis donor rats and grown to passage 2 (P2). Single‐level bilateral posterolateral intertransverse process lumbar fusion surgery was performed on syngeneic rats divided into three experimental groups: clinical‐grade bone graft substitute alone (CBGS); CBGS+ rat ASCs (rASC); and, CBGS+ rat BMCs (rBMC). Eight weeks postoperatively, fusion was evaluated via micro‐CT, manual palpation and histology. In vitro analysis of the osteogenic capacity of rBMCs and rASCs was also performed. Results indicated that the average fusion volume in the rASC group was the largest and was significantly larger than the CBGS group. Although the rASC group displayed the highest fusion rates via micro‐CT and manual palpation, this difference was not statistically significant. Cell‐seeded grafts showed more histological bone formation than cell‐free grafts. P2 rASCs and rBMCs displayed similar in vitro osteogenic differentiation capacities. Overall, this study showed that, when combined with a clinical‐grade bone graft substitute in a rat model, rASCs cells yielded the largest fusion masses and comparable fusion results to rBMCs. These results add to growing evidence that ASCs provide an attractive alternative to BMCs for spinal fusion procedures.

11550 Background: RPS often have local recurrence (LR) after surgery. Preoperative radiation (RT) to 50.4 Gy can reduce LR risk but is not uniformly effective, especially after (+) margin resections. Therefore, we conducted a multi-institutional, prospective Phase II study to assess efficacy and tolerability of preop IMPT with selective dose escalation to 63 GyRBE to the posterior RPS margin (clinical target volume [CTV] 2) at high risk for (+) margins to further reduce the risk of LR. This dose was tolerable in a prior phase I study (DeLaney T et al, 2017, PMID:28740917). Methods: Primary RPS patients (pts) >18 years received preop IMPT, 50.4 GyRBE in 28 fractions (fx) of 1.8 GyRBE to CTV1 (tumor plus adjacent tissue at risk of subclinical disease) with SIB to CTV2 to 63.0 GyRBE in 28 fx of 2.25 GyRBE. Pts with high-grade tumors could get chemotherapy(CTX) prior to IMPT. To avoid treatment delay, 11 fx of IMRT x-rays could be substituted for IMPT. Pts had restaging and surgery 4-8 weeks after IMPT. Primary study endpoint was local tumor control. Secondary endpoints included clinical and pathologic response, surgical margin status, and disease-free and overall survival. Results: We accrued 60 pts from January 2016 to February 2021. Histology: 35 liposarcoma(LPS) (19 dediff and 16 well diff), 22 leiomyosarcoma(LMS), and 3 undifferentiated pleomorphic sarcoma. IMPT was delivered per protocol in all pts. 51 pts have had surgery, 5 are awaiting surgery, and 4 had no surgery due to metastases(DM) on preop imaging. 22 pts had (+) margins. 2 pts had > 75% necrosis. With 23-month median (range 1-52 months) follow-up after start of RT, there were two LRs. A dediff LPS pt had a well diff LPS LR 26 months postop, resected, and is disease-free. A renal vein/IVC LMS pt treated with CTX and IMPT had LR and DM 4 months postop and died from disease. Surgical Clavien-Dindo morbidity scores: 0(21), 1(9), 2(8), 3a (4), 3b(4), 4a(2), 4(b)1, 5(2); the periop deaths were from sepsis(pneumonia) and duodenal ulcer. The grade 3-4 periop morbidity included abscess(3), treated by catheter(2) or operative(1) drainage, prolonged hospital stays (2 pts with IVC LMS), small bowel obstruction (1), and late sigmoid colon anastomotic failure (1). Readmissions for lymphopenia(1), pneumoperitoneum (1), and volume overload (1). One late neuropathy was seen in a Type II diabetic pt with transient postop weakness after femoral nerve dissection who later had significant lower extremity weakness 3.75 years postop. Study was amended to reduce IMPT dose in diabetic pts. Conclusions: Preoperative IMPT with selective dose escalation to 63 GyRBE to the high risk posterior RPS margin is feasible. Early local control results with this approach appear promising. Some peri-operative morbidity was noted but appears to be in the expected range for RPS resections. Clinical trial information: NCT01659203.