Response by Cui et al to Letter Regarding Article, “RGC-32 (Response Gene to Complement 32) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis”

We welcome the letter from Rus et al commenting on our recent study showing that response gene to complement 32 (RGC-32) mediates the development of atherosclerosis by facilitating monocyte-endothelial cell (EC) interaction through induction of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1.¹ Our conclusion is based on several observations: 1) RGC-32 is induced in endothelial cells in both human and mouse atherosclerotic lesions; 2) RGC-32 deficiency (Rgc32−/−) attenuates the spontaneously-developed and high fat diet (HFD)-induced atherosclerosis in Apoe−/− mice; 3) Rgc32−/− mice transplanted with wild-type (WT) bone marrow does not significantly alter the Rgc32−/− phenotype; 4) Rgc32−/− inhibits endothelial ICAM-1 and VCAM-1 expression and monocyte-endothelial cell interaction.¹ These data strongly suggest that endothelial RGC-32 plays an essential role in atherosclerosis.

We understand the concern in the letter about the differences in RGC-32 expression in human atherosclerotic lesions in our and Dr. Vlaicu et al’s studies and appreciate the insights that the discrepancy may be the result of the differences in the atherosclerotic lesions from different arterial regions. By revisiting the relevant figures in both studies,^1,2 we have realized that the discrepancy could also be caused by different immunohistochemistry staining procedures such as primary antibody concentration, secondary antibody, or substrate incubation time, etc. We have attempted to not over-stain the tissue sections in order to avoid non-specific staining, and we could observe RGC-32 staining in smooth muscle cells (SMC) and/or other cell types if our incubation times were prolonged, or greater amounts of antibodies were used. However, the results from both Vlaicu et al’s and our studies show that ECs exhibit the strongest RGC-32 staining among cells in human lesions, consistent with our conclusion that RGC-32 is predominantly, “but not only”, expressed in ECs.^1,2

We reported that RGC-32 promotes injury-induced vascular remodeling and mediates platelet-derived growth factor (PDGF)-BB-induced SMC proliferation and migration by enhancing p34^CDC2 activity and inducing focal adhesion contact and stress fiber formation, respectively.³ Our results are indeed consistent with Badea et al’s findings that RGC-32 mediates C5b-9-induced migration and proliferation of SMCs.⁴ However, SMC functions are much more complicated in atherosclerosis than mechanic injury-induced vascular remodeling. In addition to the proliferation and migration, SMCs may become macrophage-like cells and foam cells in early stage lesions, which loss the SMC marker expression.⁵ It is possible that RGC-32 promotes both SMC proliferation/migration (although with a low level of expression relative to ECs) and the SMC-macrophage/foam cell transition during the development of atherosclerosis, resulting in similar SMC and collagen contents in the lesions of Apoe−/− and Apoe−/−;Rgc32−/−mice, as observed in our study. Therefore, a future investigation is necessary to clarify the role of RGC-32 in SMC proliferation/migration and SMC-macrophage transition during the lesion development using lineage-tracking approach combined with SMC-specific RGC-32 knockout animal models.

In addition to ECs and SMCs, macrophages play a critical role in the lesion development.^{6, 7} Our previous studies have shown that RGC-32 is expressed in macrophages and promotes phagocytosis and the differentiation of classically activated (M1) macrophages.^{8, 9} Tegla et al’s studies show that RGC-32 regulates T-lymphocyte proliferation.¹⁰ These results clearly reveal that RGC-32 is important for immune cell functions. Our study does not oppose the published findings because we did not alter RGC-32 expression in macrophages in our bone-marrow transplantation experiments.¹ We used WT bone marrow cells as donors to test if vascular cells RGC-32 deletion affects atherosclerosis.¹ Since RGC-32 deletion in ECs blocks the monocyte/macrophage infiltration, an initial event for the lesion development, the subsequent macrophage function cannot be revealed even with the reconstitution of the WT macrophages.¹ However, our experiments do establish the function of vascular cell RGC-32, especially EC RGC-32, in the lesion formation. In the future, the definitive role of macrophage RGC-32 in atherosclerosis could be discovered by transplanting Apoe−/− or Rgc32−/−;Apoe−/− bone marrow to lethally irradiated Apoe−/− mice and/or generating macrophage-specific RGC-32 deletion in Apoe−/− or Ldlr−/− mice followed by HFD.

Although our study has some limitations due to the use of a global knockout mouse model, the findings actually do not contradict the published results because we did not rule out the functions of SMC and immune cell RGC-32 in atherosclerosis. We focused on ECs in this study because RGC-32 is induced ‘predominantly’ (not only) in endothelial cells.¹ However, we agree with Rus et al that further investigation is warranted in order to elucidate the role of SMC, macrophage, or other immune cell RGC-32 in atherogenesis, particularly by using tissue-specific RGC-32 deficient mouse models.