Calpastatin Counteracts Pathological Angiogenesis by Inhibiting Suppressor of Cytokine Signaling 3 Degradation in Vascular Endothelial Cells

Abstract

Rationale: Janus kinase/signal transducer and activator of transcription (JAK/STAT) signals and their endogenous inhibitor, suppressor of cytokine signaling 3 (SOCS3), in vascular endothelial cells (ECs) reportedly dominate the pathological angiogenesis. However, how these inflammatory signals are potentiated during pathological angiogenesis has not been fully elucidated. We suspected that an intracellular protease calpain, which composes the multifunctional proteolytic systems together with its endogenous inhibitor calpastatin (CAST), contributes to the JAK/STAT regulations. Objective: To specify the effect of EC calpain/CAST systems on JAK/STAT signals and their relationship with pathological angiogenesis. Methods and Results: The loss of CAST, which is ensured by several growth factor classes, was detectable in neovessels in murine allograft tumors, some human malignant tissues, and oxygen-induced retinopathy lesions in mice. EC-specific transgenic introduction of CAST caused downregulation of JAK/STAT signals, upregulation of SOCS3 expression, and depletion of vascular endothelial growth factor (VEGF)-C, thereby counteracting unstable pathological neovessels and disease progression in tumors and oxygen-induced retinopathy lesions in mice. Neutralizing antibody against VEGF-C ameliorated pathological angiogenesis in oxygen-induced retinopathy lesions. Small interfering RNA–based silencing of endogenous CAST in cultured ECs facilitated μ-calpain–induced proteolytic degradation of SOCS3, leading to VEGF-C production through amplified interleukin-6–driven STAT3 signals. Interleukin-6–induced angiogenic tube formation in cultured ECs was accelerated by CAST silencing, which is suppressible by pharmacological inhibition of JAK/STAT signals, antibody-based blockage of VEGF-C, and transfection of calpain-resistant SOCS3, whereas transfection of wild-type SOCS3 exhibited modest angiostatic effects. Conclusions: Loss of CAST in angiogenic ECs facilitates μ-calpain–induced SOCS3 degradation, which amplifies pathological angiogenesis through interleukin-6/STAT3/VEGF-C axis.

Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing

“…Cellular movements. Measurement of cellular movements was described previously (34). Twenty thousand BMMs suspended in minimal essential medium were seeded on a 24-well culture plate.…”

Recent advance in molecular angiogenesis in glioblastoma: the challenge and hope for anti-angiogenic therapy

“…Table 1 Recently discovered pro-and anti-angiogenesis factors Pro-angiogenesis TGLI1 [63], H19 [51], C/EBPbeta [14], MCSF [15], annexin A2 [39], CXCR4 [66], Oct-3/4 [16], miR-31 [50], miR-148a, HSP47 [38] Anti-angiogenesis hHSS1 [29], NNC55-0396 [30], TTAC-0001 [67], calpastatin [68], miR-137 [65], EZH2 [65], BMP7v [31], PDCD10 [32], JSI-124 [33] Brain Tumor Pathol…”

Promoting vascular repair in the retina: can stem/progenitor cells help?

“…Infiltration of microglia and monocytes/macrophages has been linked to neovascularization in OIR,25–27 and is also correlated with upregulation of proangiogenic and proinflammatory molecules, including metalloproteinases, FGF, and cytokines (ie, TNFα) 28–30. In parallel with these observations, inflammation is actually a key difference between physiological and pathological angiogenesis 31. In fact, many types of proinflammatory cytokines, including TNFα and IL-6, exert angiogenic effects on the vascular endothelium via the JAK–STAT pathway 31.…”