The protein CARD8 regulates several inflammatory proteins in people with atherosclerosis. This is shown in a new study by Örebro researchers published in the scientific journal Scientific Reports.
“We have shown entirely new functions associated with CARD8. These may be important in the future treatment of atherosclerosis and other inflammatory diseases,” says Örebro researcher Geena Paramel.
Atherosclerosis, also known as hardening of the arteries is the primary cause of most cardiovascular diseases. The inflammation in blood vessel walls caused by atherosclerosis involves several different proteins, which in turn are regulated by the protein CARD8.
“Our study shows that CARD8 plays a crucial role in the inflammatory process in atherosclerosis,” says Geena Paramel, one of the researchers behind the study and senior lecturer in biomedicine at Örebro University.
The research team, led by Professor Allan Sirsjö at Örebro University’s Cardiovascular Research Centre (CVRC), examined the functions that CARD8 plays in the cells covering the inside of blood vessels.
By suppressing the CARD8 gene, they have mapped which proteins CARD8 regulates. Results show a link between high levels of CARD8 and altered levels of several other inflammatory proteins in the hardening of arteries.
These results have been confirmed in samples from a group of individuals with atherosclerosis – in collaboration with researchers at Karolinska Institutet.
“These findings are significant in that they may be key in the development of upcoming drugs for atherosclerosis. In the future, we may be able to use more targeted drugs against CARD8 in atherosclerosis,” says Karin Franzén, docent in biomedicine at Örebro University and contributing authors in the study.
In a previous study, researchers have also seen a link in people who have a genetic variation in the CARD8 gene and altered levels of inflammatory proteins in the body.
“There is much that suggests that CARD8 may also be significant for several other inflammation-related diseases,” says Geena Paramel.
Identify the mechanism
Örebro researchers are now planning a continued CARD8 study. In collaboration with BioReperia, a company in Linköping, they will continue mapping the role that CARD8 plays in inflammatory processes that are also central to tumor development.
“Right now, we know that CARD8 plays a crucial role in the inflammatory process in cells. We also want to understand how CARD8 affects other processes in cells,” Karin Franzén explains.
Inflammation is a key component in the pathophysiology of atherosclerosis and involves numerous complex inflammatory cascades contributing to the inflammatory milieu in the atherosclerotic lesions. The caspase recruitment domain (CARD) was initially identified as a protein–protein interaction motif in the regulation of apoptosis1 and is also known for its function as scaffolding molecule to induce inflammation by activating NF-κB2,3.
During the past decade, several CARD containing proteins, such as Nod1, Nod2, CARD10, Bcl10, CARD11 and CARD14 have been identified, to regulate activation of NF-κB3 via association with different adaptor proteins; CARD6, NOD1 and NOD2 recruit RIPK2, whereas CARD9, CARD10, CARD11 and CARD14 requires recruitment of BCL10 to activate NF-κB4–9.
One of the CARD containing proteins that has previously attracted focus is CARD8 (also known as TUCAN/CARDINAL/NDDP1). The CARD8 has in earlier studies been associated with a possible role in the NLRP3 inflammasome complex and has been found over-expressed in atherosclerotic lesions10.
The CARD8 gene has been extensively studied in relation to the C10X polymorphism, and several studies have suggested a possible association with various inflammatory diseases, although ambiguous results exist11. In cardiovascular disease (CVD), the minor variant of the C10X polymorphism has previously been associated with lower expression of CARD8 mRNA levels in atherosclerotic plaques and to lower levels of CRP and MCP-1 in serum, indicating that CARD8 may aggravate the atherosclerotic process by promoting inflammation10.
However, the CARD8 regulatory mechanism is still not well understood. Unlike the other CARD proteins, CARD8 has been shown to inhibit NF-κB activation by interacting with IκB kinase complex (IKKγ)12. The CARD8 protein has also been suggested to inhibit the NOD2 dependent inflammatory response in epithelial cells and NLRP3 dependent IL-1β release in human monocyte derived macrophages13,14.
On the other hand, earlier studies showed that CARD8 does not affect the IL-1β production and release in aortic smooth muscle cells (AoSMCs)15. Due to the fact that CARD8 is overexpressed in plaque, we hypothesize that CARD8 is important for the immune modulation in atherosclerosis. The aim of the present study is to examine the expression of CARD8 in human atherosclerotic lesion and to elucidate the role of CARD8 in the regulation of inflammatory proteins in endothelial cells and atherosclerotic lesions.
In this study, we show that CARD8 protein is expressed in endothelial cells and SMCs of healthy and atherosclerotic vessels and that CARD8 may be an upstream regulator of several inflammatory cytokines and chemokines in both endothelial cells and human carotid plaques.
In a pilot study, we found that CARD8 is expressed in a variety of tissues such as brain, blood vessels, spleen, muscle, epidermis layer and hair follicles of skin (data not shown). The expression of CARD8 in endothelial cells was confirmed in microarray data generated from the Oncomine 4.4 databank (www.oncomine.org; data not shown).
In the present investigation, we found expression of CARD8 protein in endothelial cells and SMCs in non-atherosclerotic vessels. We also identified CARD8 protein expression in the intimal region in SMCs and CD68 positive cells. Previously we showed that CARD8 mRNA expression was elevated in atherosclerotic lesions10.
The present data complements the previous study by showing that in atherosclerotic lesions, immune cells, such as CD68 positive cells, endothelial cells and SMCs are the predominant cell types expressing CARD8 protein. We further correlated the expression of CARD8 in the human atherosclerotic lesion with the endothelial and macrophage markers with microarray data from the BiKE study.
Consistent with the immunostaining, the expression of CARD8 mRNA positively correlated with the mRNA expression of vWF (endothelial marker), and macrophage marker, CD163 in atherosclerotic lesions. The expression of CARD8 was localized both in the nucleus and cytoplasm of HUVECs, which was supported by a previous study where a similar subcellular localization of CARD8 was found in MCF-7 cells transfected with GFP tagged CARD8 plasmid12.
Our results are consistent with previously published analysis of gene expression pattern in atherosclerotic lesions showing that CARD8 is upregulated in human atherosclerotic lesions and is significantly correlated to genes involved in inflammatory response, including CCL2/MCP1 and CD68 suggesting a role of CARD8 in mediating inflammatory markers by macrophages in the atherosclerotic lesion16.
Knowledge about the role of CARD8 in the regulation of inflammatory markers in endothelial cells and atherosclerosis is however limited. The CARD8 belongs to the bipartite CARDs, which consists of a CARD motif and one additional motif17. In contrast to other CARD containing proteins, initial studies showed that CARD8 is an inhibitor of NF-κB activation via physical interaction with the IκB kinase complex12.
Previously, CARD8 was shown to regulate IL-1β release upon LPS and ATP stimulation in human-monocyte derived macrophages13. Contradictory, we have earlier shown that CARD8 does not affect the IL-1β levels in AoSMCs15. Furthermore, knock down of CARD8 did not affect NF-κB activation in HUVECs (data not shown).
In order to elucidate the role of CARD8 as a regulator of inflammation, we therefore silenced the CARD8 gene to elucidate the role of CARD8 in endothelial cells. Knockdown of CARD8 in HUVECs altered several proteins involved in inflammation and chemotaxis, such as CXCL1, IL-6, CXCL6, MCP-1, and PDGF-A.
This was supported by the correlation of CARD8 expression to CXCL1, PDGFA and CXCL6 and a weak positive correlation to MCP-1 and IL6 in human atherosclerotic lesions in the BiKE study, which suggests a role of CARD8 in the regulation of these proteins in human atherosclerotic plaque.
Among the several CARD8 regulated proteins identified in the present study, there are several indications that CARD8 plays a role in atherosclerosis via its impact on downstream target genes. Knockdown of the chemokine CXCL1 has in previous studies been shown to reduce atherosclerosis in atherosclerosis-prone LDLR−/− mice18.
Furthermore, CXCL6 is involved in recruitment of neutrophils, but its role in CVD remains to be elucidated19. MCP-1 is a major chemotactic protein, that acts via CCR2 receptor and is induced by modified LDL and triggers adhesion of monocytes to the endothelium20.
In the ApoE knock out mouse model, deletion of MCP-1 leads to the reduction in atherosclerotic lesion size21, which may indicate on an important regulatory role for CARD8 in atherosclerosis via MCP-1. This is also consistent with our previous study showing elevated mRNA expression in atherosclerotic lesions and that lower expression of the truncated CARD8 rare variant is associated with lower levels of MCP-1 in plasma from patients with myocardial infarction10.
Moreover, CARD8 regulates the expression of IL-6 in the endothelial cells. The cytokine IL-6 is known to regulate the synthesis of acute phase proteins, thereby contributing to the development of auto immune and chronic inflammatory disease22. IL-6 is also shown to induce the MCP-1 via JAK/STAT3 and PI3K/AKT pathways in human vascular endothelial cells23.
Several studies indicate that blocking IL-6 signaling can be protective against inflammation24. Therefore, elevated levels of CARD8 in atherosclerosis may therefore contribute to increased inflammation in the pathogenesis of atherosclerosis by upregulation of CXCL1, CXCL6, IL-6 and possibly also MCP-1, may aggravate atherosclerosis.
In the present investigation, we also found upregulation of PDGF-A after knock down of CARD8 in HUVECs and negative correlation to CARD8 mRNA expression levels in atherosclerotic lesions in the BiKE study. Elevated levels of PDGF-A have been found in atherosclerotic lesions in ApoE mice25.
In addition, PDGF-A is induced by oxidized low density lipoproteins and sheer stress in vascular smooth muscle cells and HUVECs respectively26,27. However, the role of CARD8 in the regulation of PDGF-A in atherosclerosis remains to be elucidated.
Several additional proteins were found reduced as a consequence of CARD8 knock-down, such as IL-8, TRAIL-R2, PTX3, CCL20, and MCP-3 and are involved in inflammation. Moreover, proteins like IL-8, PTX3, LDL-receptor and CCL20 are known to contribute to the development of atherosclerosis28–30.
Our study also implicates on additional roles of CARD8, since proteins of hemostasis and wound healing, such as PAR-1, TM, tPA, uPA, TFF3 and AXL were found upregulated upon silencing of CARD8, suggesting the role of CARD8 to maintain hemostasis and wound healing process.
CARD8 was also found to regulate the expression of cathepsin Z (CTSZ), a cysteine protease with exopeptidase activity regulating adhesion, migration and maturation of immune cells31. Similar to CARD8, the expression of CTSZ is significantly upregulated in human atherosclerotic lesion and was previously shown to significantly correlate to inflammatory markers, including CCL2/MCP1 and CD68 suggesting the possible role of CTSZ in inflammation16.
In conclusion, the current study shows that CARD8 protein is upregulated in atherosclerotic lesions and our data suggest that CARD8 may be involved in the regulation of the expression of cytokines and chemokines, such as CXCL1, CXCL6 and PDGF-A in vascular cells and atherosclerotic plaque, but also to other proteins related to inflammation, such as MCP-1 and IL-6.
However, additional studies are warranted to elucidate the more precise mechanism of regulation of these proteins. Although we lack in vivo evidence of CARD8 as a regulator of inflammatory markers, the present study suggest a possible role for CARD8 as a novel mediator of inflammation in atherosclerotic lesions.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644683/
More information: Geena V. Paramel et al. Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells, Scientific Reports (2020). DOI: 10.1038/s41598-020-73600-4