Approaches to reduce excessive edema due to the microvascular hyperpermeability that occurs during ischemia-reperfusion (I/R) are needed to prevent muscle compartment syndrome. We tested the hypothesis that cAMP-activated mechanisms actively restore barrier integrity in postischemic striated muscle. We found, using I/R in intact muscles and hypoxia-reoxygenation (H/R, an I/R mimic) in human microvascular endothelial cells (HMVECs), that hyperpermeability can be deactivated by increasing cAMP levels through application of forskolin. This effect was seen whether or not the hyperpermeability was accompanied by increased mRNA expression of VEGF, which occurred only after 4 h of ischemia. We found that cAMP increases in HMVECs after H/R, suggesting that cAMP-mediated restoration of barrier function is a physiological mechanism. We explored the mechanisms underlying this effect of cAMP. We found that exchange protein activated by cAMP 1 (Epac1), a downstream effector of cAMP that stimulates Rap1 to enhance cell adhesion, was activated only at or after reoxygenation. Thus, when Rap1 was depleted by small interfering RNA, H/R-induced hyperpermeability persisted even when forskolin was applied. We demonstrate that) VEGF mRNA expression is not involved in hyperpermeability after brief ischemia, ) elevation of cAMP concentration at reperfusion deactivates hyperpermeability, and) cAMP activates the Epac1-Rap1 pathway to restore normal microvascular permeability. Our data support the novel concepts that ) different hyperpermeability mechanisms operate after brief and prolonged ischemia and) cAMP concentration elevation during reperfusion contributes to deactivation of I/R-induced hyperpermeability through the Epac-Rap1 pathway. Endothelial cAMP management at reperfusion may be therapeutic in I/R injury. Here, we demonstrate that ) stimulation of cAMP production deactivates ischemia-reperfusion-induced hyperpermeability in muscle and endothelial cells;) VEGF mRNA expression is not enhanced by brief ischemia, suggesting that VEGF mechanisms do not activate immediate postischemic hyperpermeability; and ) deactivation mechanisms operate via cAMP-exchange protein activated by cAMP 1-Rap1 to restore integrity of the endothelial barrier.

Activin like kinase-1 (ALK-1) mediates signaling via the transforming growth factor beta (TGFβ) family of ligands. ALK-1 activity promotes endothelial proliferation and migration. Reduced ALK-1 activity is associated with arteriovenous malformations. No studies have examined the effect of global ALK-1 deletion on indices of cardiac remodeling. We hypothesized that reduced levels of ALK-1 promote maladaptive cardiac remodeling. Methods We studied ALK-1 conditional knockout mice (cKO) harboring the ROSA26-CreER knock-in allele whereby a single dose of intraperitoneal tamoxifen triggered ubiquitous Cre-recombinase mediated excision of floxed ALK-1 alleles. Tamoxifen treated wild-type (WT-TAM; n=5) and vehicle treated ALK-1-cKO mice (cKO-CON; n=5) served as controls for tamoxifen treated ALK-1-cKO mice (cKO-TAM; n=15). Results ALK-1 cKO-TAM mice demonstrated reduced 14-day survival compared to cKO-CON controls (13% vs 100%, respectively, p<0.01). Seven days after treatment, cKO-TAM mice began to exhibit reduced left ventricular (LV) fractional shortening, progressive LV dilation, and gastrointestinal bleeding. After 14 days total body mass was reduced, but LV and lung mass increased in cKO-TAM not cKO-CON mice. Peak LV systolic pressure, contractility, and arterial elastance were reduced, but LV end-diastolic pressure and stroke volume were increased in cKO-TAM, not cKO-CON mice. LV ALK-1 mRNA levels were reduced in cKO-TAM, not cKO-CON mice. LV levels of other TGFβ-family ligands and receptors (ALK5, TBRII, BMPRII, Endoglin, BMP7, BMP9, and TGFβ1) were unchanged between groups. Cardiomyocyte area and LV levels of BNP were increased in cKO-TAM mice, but LV levels of β-MHC and SERCA were unchanged. No increase in markers of cardiac fibrosis, Type I collagen, CTGF, or PAI-1, were observed between groups. No differences were observed for any variable studied between cKO-CON and WT-TAM mice. Conclusion Global deletion of ALK-1 is associated with the development of high output heart failure without maladaptive remodeling. Future studies exploring the functional role of ALK-1 in cardiac remodeling independent of systemic AVMs are required.

Introduction Activin receptor-like kinase 1 (ALK1) mediates signaling via the transforming growth factor beta-1 (TGFβ1), a pro-fibrogenic cytokine. No studies have defined a role for ALK1 in heart failure. Hypothesis We tested the hypothesis that reduced ALK1 expression promotes maladaptive cardiac remodeling in heart failure. Methods and Results In patients with advanced heart failure referred for left ventricular (LV) assist device implantation, LV Alk1 mRNA and protein levels were lower than control LV obtained from patients without heart failure. To investigate the role of ALK1 in heart failure, Alk1 haploinsufficient (Alk1+/−) and wild-type (WT) mice were studied 2 weeks after severe transverse aortic constriction (TAC). LV and lung weights were higher in Alk1+/− mice after TAC. Cardiomyocyte area and LV mRNA levels of brain natriuretic peptide and β-myosin heavy chain were increased similarly in Alk1+/− and WT mice after TAC. Alk-1 mice exhibited reduced Smad 1 phosphorylation and signaling compared to WT mice after TAC. Compared to WT, LV fibrosis and Type 1 Collagen mRNA and protein levels were higher in Alk1+/− mice. LV fractional shortening was lower in Alk1+/− mice after TAC Conclusions Reduced expression of ALK1 promotes cardiac fibrosis and impaired LV function in a murine model of heart failure. Further studies examining the role of ALK1 and ALK1 inhibitors on cardiac remodeling are required.

Materials/Methods: Abscopal assays were conducted in immunocompetent mice. Anti-PD-1 sensitive or resistant lung tumor cell lines were injected in both flanks. Intratumoral injection of NBTXR3 (or vehicle) followed by RT was performed in right flank (primary) tumors only. Some mice also received anti-PD-1 injections. Tumor growth was monitored, and tumor immune cell infiltrates analyzed by immunohistochemistry (IHC). Separately, in the phase II/III randomized Act.in.Sarc [NCT02379845] trial patients with locally advanced soft tissue sarcoma (STS) received either NBTXR3+RT or RT alone followed by tumor resection. Pre-and posttreatment tumor samples from patients in both groups were analyzed by IHC and Digital Pathology for immune biomarkers. The safety and efficacy of NBTXR3 plus stereotactic body radiotherapy (SBRT) in combination with anti-PD-1 is being evaluated in three cohorts of patients with advanced cancers in the Phase I 1100 [NCT03589339] trial. Results: Pre-clinical studies demonstrated that NBTXR3+RT induces an immune response not observed with RT alone and enhances systemic control. IHC showed significant increase of CD8+ T-cell infiltrates in both NBTXR3+RT treated and untreated tumors compared to RT alone. Increased CD8+ T-cell and decreased FOXP3+ Treg density (pre-vs posttreatment) was also observed in tumors from STS patients treated with NBTXR3+RT. Furthermore, NBTXR3+RT in combination with anti-PD-1 improved local and systemic control in mice bearing anti-PD-1 resistant lung tumors, produced long-term memory, and reduced spontaneous lung metastases. Preliminary efficacy data from the 1100 trial showed tumor regression in 8/9 patients. Of note, tumor regression was observed in 6/7 patients who had progressed on prior anti-PD-1. Conclusion:The clinical efficacy of NBTXR3+RT has been demonstrated as a single agent in STS. Here we demonstrate that it overcomes resistance to anti-PD-1 treatment mechanisms in mice and led to tumor regression in patients having progressed on anti-PD-1 therapy. These results highlight the potential of NBTXR3+RT to positively impact the immuno-oncology field.

Introduction: Radiation therapy (RT) is used in the treatment of approximately 50% of cancer patients. Thoracic RT is commonly used in breast cancer, Hodgkin’s lymphoma, head and neck and lung cancers among others. Unfortunately, the benefits of RT are often out-weighed by the by-stander cardiovascular risks and associated non-relapse mortality in cancer survivors. Hence, the development of cardiovascular disease (CVD) in patients receiving RT remains an important clinical problem. The base of the heart has been shown to be dose-sensitive wherein basal irradiation is associated with lower patient survival in lung-cancer patients treated with curative-intent RT. Although the mechanisms for RT-induced CVD are poorly understood, direct injury to the coronary arteries endothelium has been implicated. Hypothesis and Results: We hypothesized that irradiation harms heart tissue differentially depending upon the specific region that receives radiation. In order to test this hypothesis, we irradiated different regions of the heart and measured the development of atherosclerotic plaques as compared to control unirradiated mice. Apolipoprotein E knockout (ApoE -/- ) mice were irradiated with 16Gy to the base, apex or to the whole heart. Our results demonstrate that the base of the heart has significantly greater sensitivity to irradiation as compared to the apex, and develops atherosclerotic lesions that are equivalent to those produced by whole heart irradiation at 20 weeks post-RT. In addition, a greater number of plaques are present in the base of the heart after whole heart and basal irradiation compared to unirradiated controls at 40 weeks post-RT. Atherosclerotic plaques with subendothelial macrophage foam cell accumulation and fibrin deposits were also observed at 20 and 40 weeks post basal or whole heart RT. Ongoing studies are testing the effect of high-fat diet on the development of differential RT-induced atherosclerotic plaques in ApoE -/- mice and their correlation with heart function. Conclusion: Our studies strengthen the argument for advanced RT techniques to shield the base of the heart from radiation exposure to minimize RT-induced CVD and improve the quality of life and survival of cancer patients receiving RT.