Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w

Oxidative stress-induced, TRPC-channel mediated-RhoA kinase-ROCK axis impairs VEGFR1 signalling in hyperglycemic monocytes through PTEN phosphatase stabilisation

M. Dorenkamp1, S. Kallipatti Shanmuganathan1, D. Semo1, M. Schwietzer1, C. Adam1, I. Rubio2, H. Reinecke1, J. Waltenberger3, R. Godfrey1

1Klinik für Kardiologie I: Koronare Herzkrankheit, Herzinsuffizienz und Angiologie, Universitätsklinikum Münster, Münster; 2Klinik für Anästhesiologie und Intensivtherapie, Universitätsklinikum Jena, Jena; 3Diagnostisches und therapeutisches Herzzentrum, Hirslanden Klinik im Park, Zürich, CH;

Purpose: Monocytes play a vital role in the biological process, increasing the diameter of the existing arterial vessels. This process, also known as arteriogenesis, is essential for maintaining vascular integrity. Circulating monocytes are recruited to the sites of collateral growth where arteriogenesis is mediated through VEGFR1 signalling pathways, among others. The impaired monocyte function in hyperglycaemia — attributed to the reduced ability of monocytes to respond to VEGF stimulation — has been implicated in reduced arteriogenesis in diabetic patients. Molecular mechanisms leading to this VEGF-specific signal transduction defect in monocytes are incompletely understood. Improving monocyte function in diabetic patients may therefore have a positive effect on arteriogenesis. Methods: Human monocytes from non-T2DM or from T2DM patients were isolated from peripheral blood through gradient centrifugation and negative immunological magnet isolation. Monocyte cell line THP-1 and primary monocytes isolated from healthy donors were subjected to normoglycaemic or hyperglycaemic conditions for 7 days and 48 hours, respectively. Expression of relevant molecules was detected by RT-qPCR and confirmed by Western blotting. GST-tagged Rhotekin was used to pull down RhoA-GTP. For PTEN activity assays, immunoprecipitated PTEN was used to dephosphorylate phospho-PIP3, and the released phosphate was measured using malachite green. Inhibition of RhoA downstream kinase ROCK and transient receptor potential canonical (TRPC) channels were done using pharmacological inhibitors and transient knockdown using siRNA. VEGF-A and PlGF-1-induced monocyte chemotaxis was assessed in the modified Boyden chamber assay. Results: RT-qPCR analysis indicated an upregulation of TRPC3 and TRPC6 channels in hyperglycemic monocytes and monocytes from T2DM patients. TRPC channel upregulation was ROS-dependent as a specific NADPH oxidase inhibitor, VAS2870, blocked TRPC channel expression and calcium influx. RhoA GTPase was significantly activated in hyperglycemic monocytes and could be blocked by the pharmacological inhibition of TRPC channels. The catalytic activity of PTEN was significantly higher in hyperglycemic monocytes. Co-immunoprecipitation experiments revealed that PTEN and ROCK1 physically interact, resulting in enhanced PTEN stability. PlGF-1 stimulation of hyperglycemic monocytes does not result in AKT activation, while blockage of ROCK1 improves VEGFR1-mediated AKT signalling. Most importantly, blockage of the RhoA-ROCK axis could circumvent the attenuated response of monocytes to PlGF-1 and VEGF-A. Conclusions: Our results reveal the importance of the RhoA-ROCK axis as a negative regulator of human monocyte function in diabetic conditions. Our results suggest that the hyperglycaemia-induced TRPCchannel-mediated RhoA-ROCK activation leads to enhanced PTEN stability by direct physical interaction, thereby blocking the ligand-induced activation of AKT pathway. This results in defective monocyte chemotaxis towards PLGF-1 or VEGF-A. The RhoA-ROCK axis blockage can positively impact monocyte function by restoring VEGFR-1-AKT signalling.

https://dgk.org/kongress_programme/jt2023/aP2235.html