Yunchao Su

Calpain-2 regulation of VEGF-mediated angiogenesis

Angiogenesis is a complex process involving endothelial cell migration, proliferation, and differentiation as well as tube formation. These processes are stimulated by a variety of growth factors such as vascular endothelial growth factor (VEGF). VEGF‐induced cytoskeletal reorganization plays a crucial role in the angiogenic processes. In the present study, we evaluated the role of calpain in VEGF‐induced angiogenesis in vitro and in vivo. Human pulmonary micro‐vascular endothelial cells (PMEC) were incubated with VEGF (10–60 ng/ml) for 2–24 h, after which we measured calpain activities, protein contents of the calpain subunits and of calpastatin, endothelial mono‐layer wound repair, tube formation, and actin cytoskeleton changes. Incubation of PMEC with VEGF resulted in dose‐ and time‐dependent increases in calpain activity and protein content of calpain‐2. VEGF did not change the protein contents of calpain‐1 and the small subunit or of calpastatin. Incubation of PMEC with a VEGF receptor blocker prevented the VEGF‐induced increase in calpain activity. Inhibition of calpain activity by siRNA directed against calpain‐2 and by overexpression of calpastatin prevented VEGF‐induced increases in actin stress fibers in endothelial cells and angiogenesis. Overexpression of calpastatin also inhibits vessel formation in subcutaneous (s.c.) matrigel plugs in mice. These results indicate that calpain mediates VEGF‐induced angiogenic effects by modulating actin cytoskeletal organization.—Su, Y., Cui, Z., Li, Z., Block E. R. Calpain‐2 regulation of VEGF‐mediated angiogenesis. FASEB J. 20, 1443–1451 (2006)

Cytoskeletal regulation of nitric oxide synthase

The three isoforms of nitric oxide synthase (NOS)—endothelial NOS (eNOS), inducible NOS (iNOS), and neural NOS (nNOS)—colocalize with the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments directly or indirectly. These colocalizations enable optimal nitric oxide production and help NOS exert their functions. The reorganization of cytoskeletal polymerization state induced by extracellular stimuli such as shear stress, hypoxia, and drugs regulates eNOS, nNOS, and iNOS. Alterations of nitric oxide production caused by cytoskeletal reorganization play an important role in physiological and pathophysiological conditions. This review focuses on recent data regarding the regulation of NOS by the cytoskeleton at transcriptional, posttranscriptional, and posttranslational levels.

Use of recombinant calpain-2 siRNA adenovirus to assess calpain-2 modulation of lung endothelial cell migration and proliferation

In this study, we developed an adenoviral vector harboring calpain-2 siRNA expression unit in which sense and anti-sense strands composing the siRNA duplex were connected by a loop and transcribed into a siRNA in porcine pulmonary artery endothelial cells (PAEC). We screened one efficient adenoviral vector Ad/si-m187 and found that Ad/si-m187 successfully exerted a gene knockdown effect on calpain-2 mRNA transcription and protein expression levels. The protein content of calpain-2 was reduced by 30%–80% in PAEC infected with Ad/si-m187 in comparison to a control adenoviral vector Ad/si-luc. The mRNA levels of calpain-2 were measured by real-time PCR and were decreased by 60%–100% and in a dose dependent manner. In correspondence to silencing calpain-2 gene expression, calpain-2 activity was decreased significantly. We further evaluated the role of calpain-2 in endothelial cell migration and proliferation. PAEC infected with Ad/si-m187 displayed impaired migration and cell proliferation in comparison to cells infected with control adenoviral vector (Ad/si-luc). These results indicate that adenoviral vector harboring calpain-2 siRNA expression unit is a valuable tool to study the biology of calpains and that calpain-2 plays an important role in lung endothelial cell migration and proliferation.

Beta-actin: a regulator of NOS-3.

β-actin is traditionally considered a structural protein that organizes and maintains the shape of nonmuscle cells, although data now indicate that β-actin is also a signaling molecule. β-actin is directly associated with nitric oxide synthase type 3 (NOS-3) in endothelial cells and platelets, and this interaction increases NOS-3 activity and the affinity of NOS-3 for heat shock protein 90 kD (Hsp90). The β-actin–induced increase in NOS-3 activity may be caused directly by β-actin, the binding of Hsp90 to NOS-3, or both. Alterations in the interaction between β-actin and NOS-3 could be caused by changes either in the availability of β-actin or in the affinity of NOS-3 for β-actin, and these alterations probably contribute to vascular complications and platelet aggregation. Studies examining the interactions between NOS-3, β-actin, and Hsp90 could potentially lead to the discovery of effective peptides for the treatment of diseases associated with impaired NOS-3 activity and nitric oxide release, such as systemic and pulmonary hypertension, atherosclerosis, and thrombotic diseases.

Acute hypoxia increases intracellular L‐arginine content in cultured porcine pulmonary artery endothelial cells

Exposure to hypoxia (0% O2) for 4–24 h resulted in increased intracellular L‐arginine content and increased activity of calpain, a calcium‐dependent neutral cysteine protease, in pulmonary artery endothelial cells. Calpain‐inhibitor I abolished the increased L‐arginine content in hypoxic cells. When endothelial cell proteins were labeled with [3H]‐L‐arginine and the cells exposed to hypoxia, we observed an increase in free [3H]‐L‐arginine and a decrease in [3H]‐L‐arginine‐labeled proteins. Once again, calpain‐inhibitor I prevented the increases in free [3H]‐L‐arginine and the decreases in [3H]‐L‐arginine‐labeled proteins in hypoxic cells. Hypoxia also inhibited the synthesis of L‐arginine‐containing proteins. Thus, the increase in intracellular L‐arginine content in hypoxic pulmonary artery endothelial cells is caused by an increase in proteolysis secondary to calpain and a decrease in protein synthesis. These results indicate that hypoxia can modulate the availability of free intracellular L‐arginine, the exclusive precursor of nitric oxide (NO) and the primary substrate of NO synthase, by affecting the synthesis and degradation of cellular proteins.

Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells

The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution.

CAT-1 as a novel CAM stabilizes endothelial integrity and mediates the protective actions of L-Arg via a NO-independent mechanism.

Interendothelial junctions play an important role in the maintenance of endothelial integrity and the regulation of vascular functions. We report here that cationic amino acid transporter-1 (CAT-1) is a novel interendothelial cell adhesion molecule (CAM). We identified that CAT-1 protein localized at cell-cell adhesive junctions, similar to the classic CAM of VE-cadherin, and knockdown of CAT-1 with siRNA led to an increase in endothelial permeability. In addition, CAT-1 formed a cis-homo-dimer and showed Ca(2+)-dependent trans-homo-interaction to cause homophilic cell-cell adhesion. Co-immunoprecipitation assays showed that CAT-1 can associate with β-catenin. Furthermore, we found that the sub-cellular localization and function of CAT-1 are associated with cell confluency, in sub-confluent ECs CAT-1 proteins distribute on the entire surface and function as L-Arg transporters, but most of the CAT-1 in the confluent ECs are localized at interendothelial junctions and serve as CAMs. Further functional characterization has disclosed that extracellular L-Arg exposure stabilizes endothelial integrity via abating the cell junction disassembly of CAT-1 and blocking the cellular membrane CAT-1 internalization, which provides the new mechanisms for L-Arg paradox and trans-stimulation of cationic amino acid transport system (CAAT). These results suggest that CAT-1 is a novel CAM that directly regulates endothelial integrity and mediates the protective actions of L-Arg to endothelium via a NO-independent mechanism.