Attenuated Joint Tissue Damage Associated With Improved Synovial Lymphatic Function Following Treatment With Bortezomib in a Mouse Model of Experimental Posttraumatic Osteoarthritis.

OBJECTIVE: To investigate the roles of the synovial lymphatic system in the severity and progression of joint tissue damage and functional responses of synovial lymphatic endothelial cells (LECs) to macrophage subsets, and to evaluate the therapeutic potential of the proteasome inhibitor bortezomib (BTZ) in a mouse model of experimental posttraumatic osteoarthritis (OA). METHODS: C57BL/6J wild-type mice received a meniscal ligamentous injury to induce posttraumatic knee OA. Lymphangiogenesis was blocked by a vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody. Synovial lymphatic drainage was examined by near-infrared imaging. Joint damage was assessed by histology. RNA-sequencing and pathway analyses were applied to synovial LECs. Macrophage subsets in the mouse synovium were identified by flow cytometry and immunofluorescence staining. M1 and M2 macrophages were induced from mouse bone marrow cells, and their effects on LECs were examined in cocultures in the presence or absence of BTZ. The effects of BTZ on joint damage, LEC inflammation, and synovial lymphatic drainage were examined. RESULTS: Injection of a VEGFR-3 neutralizing antibody into the joints of mice with posttraumatic knee OA reduced synovial lymphatic drainage and accelerated joint tissue damage. Synovial LECs from the mouse OA joints had dysregulated inflammatory pathways and expressed high levels of inflammatory genes. The number of M1 macrophages was increased in the knee joints of mice with posttraumatic OA, thereby promoting the expression of inflammatory genes by LECs; this effect was blocked by BTZ. Treatment with BTZ decreased cartilage loss, reduced the expression of inflammatory genes by LECs, and improved lymphatic drainage in the knee joints of mice with posttraumatic OA. CONCLUSION: Experimental posttraumatic knee OA is associated with decreased synovial lymphatic drainage, increased numbers of M1 macrophages, and enhanced inflammatory gene expression by LECs, all of which was improved by treatment with BTZ. Intraarticular administration of BTZ may represent a new therapy for the restoration of synovial lymphatic function in subjects with posttraumatic knee OA.

Synthesis and biological profile of the pan-vascular endothelial growth factor receptor/tyrosine kinase with immunoglobulin and epidermal growth factor-like homology domains 2 (VEGF-R/TIE-2) inhibitor 11-(2-methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (CEP-11981): a novel oncology therapeutic agent.

A substantial body of evidence supports the utility of antiangiogenesis inhibitors as a strategy to block or attenuate tumor-induced angiogenesis and inhibition of primary and metastatic tumor growth in a variety of solid and hematopoietic tumors. Given the requirement of tumors for different cytokine and growth factors at distinct stages of their growth and dissemination, optimal antiangiogenic therapy necessitates inhibition of multiple, complementary, and nonredundant angiogenic targets. 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11b, CEP-11981) is a potent orally active inhibitor of multiple targets (TIE-2, VEGF-R1, 2, and 3, and FGF-R1) having essential and nonredundant roles in tumor angiogenesis and vascular maintenance. Outlined in this article are the design strategy, synthesis, and biochemical and pharmacological profile for 11b, which completed Phase I clinical assessing safety and pharmacokinetics allowing for the initiation of proof of concept studies.

Cryptotanshinone inhibits lymphatic endothelial cell tube formation by suppressing VEGFR-3/ERK and small GTPase pathways.

Cryptotanshinone (CPT), isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, the underlying mechanism remains to be defined. Here, we show that CPT inhibited lymphangiogenesis in an in vitro model (tube formation). This effect was partly attributed to inhibiting expression of VEGF receptor 3 (VEGFR-3) in murine lymphatic endothelial cells (LEC), as overexpression of VEGFR-3 conferred resistance to CPT inhibition of the tube formation, whereas downregulation of VEGFR-3 mimicked the effect of CPT, blocking the tube formation. Furthermore, CPT inhibited phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2). Overexpression of VEGFR-3 attenuated CPT inhibition of ERK1/2 phosphorylation, whereas downregulation of VEGFR-3 inhibited ERK1/2 phosphorylation in LECs. Expression of constitutively active MKK1 resulted in activation of ERK1/2 and partially prevented CPT inhibition of LEC tube formation. In addition, CPT also inhibited protein expression and activities of Rac1 and Cdc42 but not RhoA. Expression of constitutively active Rac1 and Cdc42 concurrently, but not Rac1 or Cdc42 alone, conferred resistance to CPT inhibition of LEC tube formation. Taken together, the results suggest that CPT inhibits LEC tube formation, in part, by inhibiting VEGFR-3-mediated ERK1/2 phosphorylation and, in part, by inhibiting expression of the small GTPases.

Inhibition of lymphangiogenesis and lymphatic drainage via vascular endothelial growth factor receptor 3 blockade increases the severity of inflammation in a mouse model of chronic inflammatory arthritis.

OBJECTIVE: This study was undertaken to investigate the effect of lymphatic inhibition on joint and draining lymph node (LN) pathology during the course of arthritis progression in mice. METHODS: Tumor necrosis factor (TNF)-transgenic mice were used as a model of chronic inflammatory arthritis. Mice were subjected to contrast-enhanced magnetic resonance imaging to obtain ankle and knee joint synovial volumes and draining popliteal LN volumes before and after 8 weeks of treatment with vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody, VEGFR-2 neutralizing antibody, or isotype IgG. Animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining popliteal LNs. Histologic, immunohistochemical, and reverse transcriptase-polymerase chain reaction analyses were used to examine lymphatic vessel formation and the morphology of joints and popliteal LNs. RESULTS: Compared with IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of popliteal LNs, the number of lymphatic vessels in joints and popliteal LNs, lymphatic drainage from paws to popliteal LNs, and the number of VEGF-C-expressing CD11b+ myeloid cells in popliteal LNs. However, it increased the synovial volume and area of inflammation in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation. CONCLUSION: These findings indicate that lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation.

Malignant mesothelioma growth inhibition by agents that target the VEGF and VEGF-C autocrine loops.

Malignant mesothelioma (MM) is a locally aggressive tumor that originates from the mesothelial cells of the pleural and sometimes peritoneal surface. Conventional treatments for MM, consisting of chemotherapy or surgery give little survival benefit to patients, who generally die within 1 year of diagnosis. Hence, there is an urgent need for the development of alternative therapies. Vascular endothelial growth factor (VEGF) is an autocrine growth factor for MM. The closely related molecule, VEGF-C, is also implicated in malignant mesothelioma growth. VEGF-C and its cognate receptor VEGFR-3 are co-expressed in mesothelioma cell lines. A functional VEGF-C autocrine growth loop was demonstrated in mesothelioma cells by targeting VEGF-C expression and binding to VEGFR-3. The ability of novel agents that reduce the levels of VEGF and VEGF-C to inhibit mesothelioma cell growth in vitro was assessed. Antisense oligonucleotide (ODN) complementary to VEGF that inhibited VEGF and VEGF-C expression simultaneously specifically inhibited mesothelioma cell growth. Similarly, antibodies to VEGF receptor (VEGFR-2) and VEGF-C receptor (VEGFR-3) were synergistic in inhibiting mesothelioma cell growth. In addition, a diphtheria toxin-VEGF fusion protein (DT-VEGF), which is toxic to cells that express VEGF receptors was very effective in inhibiting mesothelioma cell growth in vitro. These results indicate that targeting VEGF and VEGF-C simultaneously may be an effective therapeutic approach for malignant mesothelioma.