Cell Senescence and Inflammation: A Link to Atherosclerosis

Summary of:

Ito, T. K., Yokoyama, M., Yoshida, Y., Nojima, A., Kassai, H., Oishi, K., & … Minamino, T. (2014). A crucial role for CDC42 in senescence-associated inflammation and atherosclerosis. Plos One, 9(7). doi:10.1371/journal.pone.0102186


Trevor Griesman and Alissa Meister

Atherosclerosis is a chronic inflammatory disease affecting medium-large arteries beginning at birth, with the progression depending on many factors. The main risk factors for atherosclerosis include hypertension, hyperlipidemia, diabetes mellitus and others such as age, sex, smoking, and sedentary lifestyle. Possible complications from atherosclerosis include coronary artery disease, cerebrovascular diseases, and peripheral artery disease. Although these complications are responsible for over half of the yearly world mortality, they often occur late in the progression of the disease with limited early diagnoses.

The first molecular event in atherosclerosis is endothelial dysfunction in the arteries as a result of injury or inflammation. This dysfunction takes the form of either cell senescence, apoptosis, or activation. This paper focuses on senescence, when cells stop replicating. Atheroscletotic plaques result from an accumulation of lipids and smooth muscle cell proliferation. In response, endothelial cells over-express adhesion molecules and increase the recruitment of inflammatory cells. These  inflammatory cells further release cytokines, causing a cytokine-mediated progression of atherosclerosis and LDL oxidation.  Plaques deteriorate the cell wall and cause thickening of the surrounding muscles. Accumulation of these plaques can limit the flow of oxygen and nutrients to the rest of the body, leading to serious consequences depending on where these plaques form. Plaques forming in the coronary arteries cause Coronary Artery Disease (CAD), limiting the blood flow to the heart and increasing the risk for blood clots. Similar diseases can develop if these plaques form in the periphery, carotid artery, etc. Symptoms of atherosclerosis also depend on where these plaques form, and may include chest pain, weakness, numbness in the periphery, headache, kidney disease, and many more.

CDC42 is a GTPase in the Rho family responsible for cell cycle regulation functions such as migration, endocytosis, morphology, and cell-cycle progression. Additionally, CDC42 regulates the organization and proliferation of the actin cytoskeleton. Previous studies have shown that CDC42 may act in the senescence and inflammation of cells. The CDC42 pathway has been implicated as a potential therapeutic target for inflammation reduction in aging individuals, however this has yet to be studied in an in vivo model. Ito et al. (2014) set out to study the link between CDC42 and inflammation in senescent cells, and connect this to a potential role of CDC42 in atherosclerosis. The researchers specifically investigated the relationship between CDC42 and the inflammatory NF-κB pathway.



In order to create a model for senescence, the researchers cloned the genes for p16 and p21 (cyclin-dependent kinase inhibitors) into retroviral vectors, and infected cells. This resulted in the integration of the gene DNA into the DNA of the cells. In order to determine the effects of different CDC42 pathway components, they knocked down genes using siRNA. They then used RT-qPCR (as we did in class) to measure the mRNA levels of three genes: the cytokine CCL2, the endothelial-leukocyte molecule E-selectin (SELE), and Vascular Cell Adhesion Molecule 1 (VCAM1). Western blotting was used to quantify translated levels of these proteins. For some experiments, other proteins, such as a deactivated form of CDC42 were upregulated in cells by infection with an adenoviral vector which contained the DNA that coded for those proteins. To measure innate immune response levels, induced-senescent cells transfected with various siRNAs were treated with LPS or TNF-α, and levels of CCL2, SELE, and VCAM1 mRNA were measured by RT-qPCR.

The researchers also studied mouse and roundworm models. Mice were made into conditional knockouts using a Cre-Lox system, in which special sequences called lox sequences are inserted into the genome flanking the target gene. The protein Cre cuts at the lox regions. A Cre protein was used that is inactive until treated with a drug, in this case tamoxifen. When the researchers introduced tamoxifen into their Cre-Lox mice, they effectively knocked out the gene between the lox sequences. The researchers targeted either CDC42, Mdm2 (a negative regulator of p53), or some combination of those genes. They also used Apolipoprotein E knockout mice. In these mice, they investigated protein expression in tissue sections using immunostaining. They recorded mRNA levels using RT-qPCR, and measured atherosclerotic lesion areas using tissue section staining. Finally, the researchers studied C. elegans worms, specifically the nol-6 strain, which has high innate immune expression due to a p53 dependant pathway. The researchers measured levels of immunity-gene mRNA using RT-qPCR in siRNA treated worms, and tracked the survival of the worms over time.



The researchers found that in senescence-induced cells, the NF-κB pathway upregulates pro-inflammatory gene expression (Figure 1). This was shown through the creation of senescent like cells by introducing pathway kinase inhibitors, which led to an increase in inflammatory cytokine and adhesion molecule expression. Furthermore siRNA directly targeting this pathway decreased the inflammatory cytokines. The researchers additionally found that CDC42 also regulates pro-inflammatory gene expression, by testing knockdowns of the CDC42 pathway and observing a decrease in inflammatory molecules (Figure 2). After these two findings surrounding NF-κB and CDC42, the researchers further found that CDC42 up-regulates pro-inflammatory cytokines by activating NF-κB. However, when cells were activated by either LPS or TNF-α, CDC42 knockdowns had had cytokine levels higher than NF-κB knockdowns, implying that NF-κB is downstream of CDC42, as it functions with other methods of activation (Figure 3).

The researchers then decided to test these finding using in vivo models, including several strains of mice and C. elegans. An increase in pro-senescence signalling in atherosclerotic plaques was found, linking atherosclerosis and inflammation (Figure 4). Additionally, CDC42 deletion was found to reduce aortic infiltration by macrophages in atherosclerosis. Overall, using these in vivo models, the researchers found that CDC42 is a mediator of chronic inflammation, which leads to endothelial senescence.



In senescent cells, replication is halted as cells age to prevent the onset of cancer or other genetic malfunctions. Senescent cells secrete inflammatory cytokines, possibly as a way of signalling to immune cells that will destroy the senescent cell before it becomes cancerous. As we discussed in class, one of the ways that endothelial cells can respond to stress is to become senescent. This can lead to the formation of plaques, as lipids and then immune cells infiltrate the tunica intima. The researchers investigated CDC42 because of its involvement in inflammation following senescence. In the context of plaque formation, if the senescent endothelial cells did not secrete inflammatory cytokines, less immune cells would invade the plaque, slowing the progression of the plaque and possibly preventing the formation of a fibrous cap. However, clinical treatments knocking out CDC42 are very far off, as the inhibition of all senescent inflammatory signalling could result in the buildup of senescent cells, and there is no way to target CDC42 specific siRNA to plaque sites. Additionally, the experiments were done in cell models of senescence, but other factors may contribute to senescence in vivo. 




Mitrovska, S. (2009). Atherosclerosis : Understanding Pathogenesis and Challenge forTreatment. New York: Nova Biomedical Books.

What Is Atherosclerosis? (2014, August 4). Retrieved March 6, 2015, from http://www.nhlbi.nih.gov/health/health-topics/topics/atherosclerosis


Understanding the Complex Role of Isoprenoid Depletion in MKD

Summary of:

Burgh, Pervolaraki, Turkenburg, Waterham, Frenkel, & Boes. Unprenylated RhoA Contributes to IL-1b Hypersecretion in Mevalonate Kinase Deficiency Model through Stimulation of Rac1 Activity. The Journal of Biological Chemistry Vol. 289, No. 40, pp. 27757-27765, October 2014.

Hereditary autoinflammatory disorders are caused by genetic mutations in molecules involved in regulating the innate immune response. They are characterized by recurrent and usually short attacks of joint pain, rashes, abdominal pain, and fever. One category of these diseases includes hyperimmunoglobulinemia D and periodic fever syndrome (HIDS) and mevalonic aciduria (MA) and is caused by mutations in mevalonate kinase, an enzyme that acts upstream in the mevalonate pathway (Figure 1). Mevalonate kinase is responsible for the production of both non-sterol (farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) and sterol isoprenoids (cholesterol). Taken together, these disorders are referred to as mevalonate kinase deficiency (MKD). Patients with MKD suffer from periodic fever episodes, which have been attributed, at least in part, to depletion GGPP and subsequent uncontrolled release of the cytokine interleukin (IL)-1b from monocytes and macrophages.

Figure 1 (Clin Cancer Res, July 1, 2012 vol. 18 no. 13 3524-3531)


How does GGPP control IL-1b release? GGPP is a lipid moiety that is responsible for the prenylation of proteins including the Rho and Rab families of small GTPases, which cycle between a GTP bound active state and a GDP bound inactive state. Modification of these proteins by GGPP is involved in subcellular localization (membrane attachment) and the control of GTP hydrolysis via interaction with guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and GDP dissociation inhibitors (GDIs). GEFs facilitate the exchange of GDP for GTP to generate the activated form. GAPs increase the GTPase activity thereby causing inactivation. Finally, GDIs interact with the prenylated, GDP bound form to inhibit GEF/GAP binding and membrane localization (Figure 2). Lack of prenylation by GGPP has the potential to either activate (for example by blocking GDI interaction) or inhibit (for example by disrupting proper localization) GTPases depending on the circumstances.


Figure 2 (Genes & Dev. 2002, 16: 1587-1609)

In MKD cell culture models, Rac1, a member of the Rho family of GTPases, is known to have reduced prenylation by GGPP leading to increased activity, which has previously been reported to mediate IL-1b hypersecretion. However, activation of Rac1 does not account for all MKD related phenotypes and the exact mechanism of RacI activation has not been determined. This article explores the role of protein prenylation in the function of another Rho family member, RhoA, and hypothesizes that lack of RhoA prenylation contributes to some MKD phenotypes. In order to test this hypothesis, a cell culture model of MKD was used where THP-1 human moncytic cells were treated with simvastatin, a drug that inhibits the enzyme directly upstream of mevalonate kinase, HMG-CoA reductase. As previously observed, simvastatin treatment resulted in increased GTP bound Rac1, indicative of activation. However, simvastatin treatment resulted in decreased GTP-RhoA, indicative of decreased activation. In addition, inhibition of RhoA with C3 transferase (an inhibitor more specific for RhoA than simvastatin) resulted in increased GTP-bound (and therefore activated) Rac1 and IL-1b release. Taken together, these results indicate that decreased prenylation of RhoA leads to inactivation, which subsequently leads to increased Rac1 activation and IL-1b release. Ultimately, this study increased our understanding of molecular mechanisms related to MKD pathogenesis by adding another piece to the puzzle related to IL-1b hypersecretion therefore uncovering a novel target for treatment.