Thrombospondin 2 (TSP2) can inhibit angiogenesis in vitro by limiting proliferation and inducing apoptosis of endothelial cells (ECs). TSP2 can also modulate the extracellular levels of gelatinases (matrix metalloproteases, MMPs) and potentially influence the remodeling of the extracellular matrix (ECM). Here, we tested the hypothesis that by regulating MMPs, TSP2 could alter EC-ECM interactions. By using a three-dimensional angiogenesis assay, we show that TSP2, but not TSP1, limited angiogenesis by decreasing gelatinolytic activity in situ. Furthermore, TSP2-null fibroblast-derived ECM, which contains irregular collagen fibrils, was more permissive for EC migration. Investigation of the role of TSP2 in physiological angiogenesis in vivo, using excision of the left femoral artery in both TSP2-null and wild-type mice, revealed that TSP2-null mice displayed accelerated recovery of blood flow. This increase was attributable, in part, to an enhanced arterial network in TSP2-null muscles of the upper limb. Angiogenesis in the lower limb was also increased and was associated with increased MMP-9 deposition and gelatinolytic activity. The observed changes correlated with the temporal expression of TSP2 in the ischemic muscle of wild-type mice. Taken together, our observations implicate the matrix-modulating activity of TSP2 as a mechanism by which physiological angiogenesis is inhibited. The thrombospondins (TSPs) are a small family of five, secreted, modular glycoproteins (TSPs 1 to 5), with diverse functions.1,2 TSP1 and TSP2 share a high degree of similarity and are thought to constitute a subfamily. TSP1 has been extensively studied and has been shown to be synthesized by a variety of cells and to interact with a number of receptors such as CD36, CD47, GPIIb/IIIa, heparan sulfate proteoglycan, low-density lipoprotein receptor-related protein (LRP), and several integrins.3 TSP2, has not been extensively studied, but because of its similarity to TSP1 it is believed that it can bind to the same receptors. 4,5 In fact, CD36, heparan sulfate proteoglycan, LRP, and ␣ V ␤3 have been shown to be receptors for TSP2. 6,7 TSPs have also been shown to interact with several extracellular matrix (ECM) proteins including collagen, fibrinogen, and fibronectin. 2,8 Recently, the very low-density lipoprotein receptor was shown to be a receptor for TSP1 and TSP2, and their interaction was shown to inhibit the division of microvascular endothelial cells (ECs). 9TSP1 was identified as the first endogenous inhibitor of angiogenesis.10 The anti-angiogenic activities of TSP1 and TSP2 have been the focus of rigorous investigation and numerous studies have implicated both proteins in the regulation of tumor angiogenesis.3,11-13 TSP1 and TSP2 have also been shown to have broad anti-angiogenic activities in in vivo and in vitro assays, and a down-regulation of TSP1 synthesis has been implicated in a number of pathological conditions that involve increased angiogenesis. Like TSP1, TSP2 can directly influence ECs by inhibiting basic fibroblast...

Injury-and ischemia-induced angiogenesis is critical for tissue repair and requires nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS). We present evidence that NO induces angiogenesis by modulating the level of the angiogenesis inhibitor thrombospondin 2 (TSP2). TSP2 levels were higher than WT in eNOS KO tissues in hind-limb ischemia and cutaneous wounds. In vitro studies confirmed that NO represses TSP2 promoter activity. Moreover, double-eNOS/TSP2 KO mice were generated and found to rescue the phenotype of eNOS KO mice. Studies in mice with knock-in constitutively active or inactive eNOS on the Akt-1 KO background showed that eNOS activity correlates with TSP2 levels. Our observations of NO-mediated regulation of angiogenesis via the suppression of TSP2 expression provide a description of improved eNOS KO phenotype by means other than restoring NO signaling.wound healing | extracellular matrix | Akt | matrix metalloproteinases E ndothelial nitric oxide synthase (eNOS) and its bioactive product nitric oxide (NO) are well-established proangiogenic molecules. Endothelial-derived NO is crucial for maintenance of proper vasodilatory tone and regulation of an antiproliferative and antiapoptotic state for endothelial cells (ECs) and has essential roles in physiological angiogenesis (1-3). Pharmacological inhibition or genetic disruption of eNOS limited angiogenesis during tissue repair, resulting in delayed wound closure (2, 4). Addition of NO donors to wounds enhanced angiogenesis and accelerated healing (5-7). eNOS KO mice recovered poorly from hind-limb ischemia as a consequence of decreased angiogenesis (3,8). These mice also displayed accelerated atherosclerosis, neointimal thickening postinjury, and hypertension (1, 9). Taken together, these observations highlight the ability of eNOS-derived NO to influence vascular function.Thrombospondins (TSPs) are a small family of antiangiogenic matricellular proteins (10). TSPs enhance clearance of matrix metalloproteinase (MMP)-2 and MMP-9 (11-13) and interact with cell-surface receptors, including α v β 3 , very low density lipoprotein receptor (VLDLR), CD36, and CD47, to inhibit angiogenesis (14). Further, ultrastructural studies demonstrated that TSP2 influences ECM assembly (15, 16). TSP2 KO mice displayed improved recovery of blood flow following ischemia (17), altered foreign body response (18,19), and accelerated wound healing (16,20,21). In contrast, TSP1 KO mice displayed delayed healing because of insufficient stimulation of inflammation (13). Consistent with these observations, the expression of TSP1 and TSP2 in tissue repair was associated with the inflammatory and repair phases, respectively (13, 16). Recently, several studies linked components of the Akt-eNOS cascade with TSPs. Specifically, TSP1 has been described to blunt the ability of NO to activate soluble guanyl cyclase (sGC) (22, 23) through interactions with CD36 and CD47 during ischemia. TSP1 has also been described to diminish eNOS activity by blocking phosphorylation at S117...

SummaryInfections with hepatitis B virus (HBV) can initiate chronic hepatitis and liver injury, causing more than 600,000 deaths each year worldwide. Current treatments for chronic hepatitis B are inadequate and leave an unmet need for immunotherapeutic approaches. We designed virus-like vesicles (VLV) as self-amplifying RNA replicons expressing three HBV antigens (polymerase, core, and middle surface) from a single vector (HBV-VLV) to break immune exhaustion despite persistent HBV replication. The HBV-VLV induces HBV-specific T cells in naive mice and renders them resistant to acute challenge with HBV. Using a chronic model of HBV infection, we demonstrate efficacy of HBV-VLV priming in combination with DNA booster immunization, as 40% of treated mice showed a decline of serum HBV surface antigen below the detection limit and marked reduction in liver HBV RNA accompanied by induction of HBsAg-specific CD8 T cells. These results warrant further evaluation of HBV-VLV for immunotherapy of chronic hepatitis B.

Virus-like vesicles (VLV) are hybrid vectors based on an evolved Semliki Forest virus (SFV) RNA replicon and the envelope glycoprotein (G) from vesicular stomatitis virus (VSV). Previously, we showed that VLV can be used to express protein antigens and generate protective antigen-specific CD8+ T cells. This report describes VLV vectors designed for enhanced protein expression and immunogenicity. Expressing hepatitis B virus (HBV) middle S antigen (MHBs) from VLV using a dual subgenomic promoter significantly increased MHBs-specific CD8+ T cell and antibody production in mice. Furthermore, envelope glycoprotein switch from VSV Indiana to the glycoprotein of Chandipura virus enabled prime-boost immunization and further increased responses to MHBs. Therapeutic efficacy was evaluated in a mouse model of chronic HBV infection initiated by HBV delivery with adeno-associated virus. Mice with lower or intermediate HBV antigen levels demonstrated a significant and sustained reduction of HBV replication following VLV prime-boost immunization. However, mice with higher HBV antigen levels showed no changes in HBV replication, emphasizing the importance of HBV antigenemia for implementing immunotherapies. This report highlights the potential of VLV dual promoter vectors to induce effective antigen-specific immune responses and informs the further development and evaluation of hybrid viral vaccine platforms for preventative and therapeutic purposes.

Background: Ovarian cancer is the most lethal of all gynecologic malignancies with a grim 5-year survival rate of ~47%. Mortality occurs in the setting of recurrent disease wherein the co-presentation of chemoresistance and carcinomatosis limits the value of standard of care. It is therefore imperative to develop approaches that can prevent recurrent ovarian cancer. Oncolytic viruses are genetically modified replication competent viruses that can selectively infect cancer cells to induce cell lysis and death. This approach has proven to be clinically safe making it a promising approach to complement standard of care. The objective of this study is to evaluate the efficacy of CARG-2020, a virus-like vesicles (VLV) delivering three immune-modulators including IL-12, IL-17 antagonist and shRNA-PD-L1 in preventing recurrent disease in a syngeneic mouse model of recurrent ovarian cancer. Materials and Methods: In vitro: Triple knockout (TKO; p53LSL-R172H/Dicerflox/flox/Ptenflox/flox) mouse ovarian cancer cells were seeded in 96-well plates and treated with different doses of CARG-2020. Cell death was determined using the Incucyte imaging system. In vivo: TKO ovarian cancer cells stably expressing the mCherry fluorescent protein were injected i.p. in C57bl/6 mice (day 0) to mimic the establishment of recurrent disease. Treatment commenced on day 3 with the treatment group receiving three doses of 1x10^8 PFU of CARG-2020 given every other day (n=5). Control group received PBS (n=5). Both treatments were given i.p. Tumor growth was monitored by live imaging using mCherry fluorescence ROI area. Progression-free survival (PFS) was defined as the day ROI area reached 2,000. Results: CARG-2020 (1x10^5 PFU) demonstrated cytolytic effect in vitro and induced 100% cell death within 24h. In vivo, CARG-2020 induced significant decrease in i.p. tumor growth (p = 0.0128) compared to PBS control. Treatment with CARG-2020 completely delayed the establishment of recurrent disease (PFS, p = 0.0003). Whereas PBS control group demonstrated median PFS of 20 days, mice in the CARG-2020 group remained disease-free until day 60. Conclusion: CARG-2020 is able to prevent the establishment of recurrent disease in a syngeneic mouse model of recurrent ovarian cancer. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients. Citation Format: Ayesha B. Alvero, Alexandra Fox, Bhaskara Madina, Valerian Nakaar, Timur Yarovinsky, Marie Krady, Bijan Almassian, Gil Mor. CARG-2020 artificial oncolytic virus delivering three immune-modulators prevents tumor recurrence in a syngeneic model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1150.