Presentation Summary

Written by Jasna Trbojevic-Stankovic
Reviewed by Catherine Shanahan

Patients with chronic kidney disease (CKD) develop accelerated vascular calcification, predominantly of the vessel media where the entire vascular smooth muscle cell (VSMC) layer is covered in calcium and phosphate crystals, that is a major cause of cardiovascular mortality. This form is also present in patients with diabetes and in aged individuals. Another form, intimal calcification, is associated with atherosclerosis and it is a strong indicator of cardiovascular mortality, regardless of its location in the body. In the past, vascular calcification was considered a degenerative age-associated phenotype and an unmodifiable risk factor.

Mechanisms of VMSCs calcification
Calcification is a VSMC-mediated process. Normally, VMSCs are contractile and well protected against calcification by inhibitors that they either express themselves, such as matrix Gla protein, or that are in the circulation, such as fetuin-A. Diabetes, CKD, and ageing are risk factors for damaging VSMCs and dysregulating mineral metabolism, while phosphate is one of the key damaging agents. When VSMCs are damaged, they try to repair the vessel wall. If the damage is ongoing, they take on an osteogenic phenotype, orchestrate the calcification process, and start to take up nanocrystals, become osteogenic, and die in a vicious cycle [1]. Cellular ageing encompasses many processes. Firstly, DNA damage makes the cells become senescent, stop proliferating, and induce the inflammatory response in the local area. In addition, nuclear lamina defects cause multiple diseases that have tissue-specific manifestations of ageing, and together with epigenetic modifications, mitochondrial damage and proteostasis, form key processes that lead to cellular ageing.

The role of the nuclear lamina
The nuclear lamina is an intermediate filament network that underlies the inner nuclear membrane and ensures that the nuclear functions are able to undergo normal processes. The transport between the cytoplasm and the nucleus, the role of maintaining chromatin, and therefore the epigenetic profile of the cell are also regulated by the nuclear lamina as well as DNA replication and DNA repair. Mutations in the gene that encodes the nuclear lamina protein, lamin A, cause more than 18 tissue-specific diseases including Hutchinson-Gilford progeria syndrome (HGPS), which is manifested with tissue-specific mechanisms of ageing. Children affected by HGPS die of myocardial infarction or stroke by the time they are 16 years old and their vasculature is characterized by stiff calcified arteries and the loss of VSMCs. Since lamin A is made from a precursor protein, prelamin A, HGPS affected children have the mutation that deletes key amino acid sequences that are required for the final posttranslational modification. Therefore, the children accumulate the precursor protein, prelamin A, in a mutant or unprocessed form [2]. When cultured VSMCs become senescent, they have the exact nuclear morphology defects as children with HGPS. Specifically, VSMCs from old donors reached replicative senescence sooner as compared to younger donors, as seen using Western blotting. Electron microscopy shows very convoluted nuclei, as also demonstrated in HGPS children [3].

Relationship between senescence and vascular ageing
Looking at in vivo aortic samples, there was no prelamin A accumulation in younger patients as opposed to high levels of prelamin A accumulation in older calcified patients, due to downregulation of the protein that modifies prelamin A because of ageing [3]. When serial passaging of primary human VSMCs was performed in vitro, the aged VSMCs grew normally, and then stopped growing, started accumulating prelamin A and finally underwent rapid senescence. Therefore, prelamin A accumulation is driving the cells into rapid senescence. Additionally, when prelamin A is accumulated, cells start to express osteogenic genes suggesting the relationship between calcification and senescence. When prelamin A was expressed in healthy VSMCs in vitro, they rapidly underwent senescence. VMSCs upregulated P16, a key senescent marker demonstrating that these cells have exited the cell cycle. Calcifying cells also accumulated prelamin A, became senescent and upregulated P16 and other osteogenic genes [3, 4]. Therefore, prelamin A interferes with a number of DNA damage repair pathways in order to cause persistent DNA damage (Slide 13 [5]).

DNA damage response signalling pathways
Depending on to the type of DNA damage, many pathways can regulate DNA damage signalling. Two of them are poly ADP ribose polymerase (PARP) and ataxia telangiectasia, mutated (ATM). PARP is relevant to oxidative DNA damage, while ATM regulates several kinds of DNA damage repair. If the DNA repair is not successful, the cell undergoes apoptosis or senescence. In vitro, calcification may be completely blocked together with osteogenic differentiation of VSMCs by including ATM inhibitors [4]. Senescent cells release many senescence associated secretory phenotype (SASP) factors, which are growth factors, cytokines and soluble receptors that can drive ageing and dysfunction in the cells around the senescent cells.
Interestingly, many of the SASP factors are able to regulate calcification, such as interleukin 6 (IL6) and bone morphogenetic protein 2 (BMP2) [1]. Importantly, co-culture experiments with mesenchymal precursor cells showed that the increased osteogenic differentiation was promoted by paracrine effects (Slides 17, 18 [5]). It was verified by ELISA and PCR that the release of osteogenic factors by VSMCs could be blocked by ATM inhibition [4].

Premature vascular calcification in CKD patients
CKD patients have premature vascular calcification that is driving the age-associated phenotype [6-10], so that prelamin A accumulation in calcified arteries of young children on haemodialysis (HD) makes their arteries age independently of their chronological age. Their vessels have accumulated oxidative damage expressed by increased levels of 8-oxo-dG biomarker, as well as senescence markers P16 and P21, the latter of which started to accumulate in the pre-HD stage. Ex vivo treatment with calcium and phosphate showed further oxidative DNA damage in the HD children vessel rings, while the healthy vessels remained protected. In vitro testing showed that the vessels from HD children had a defective damage repair possibly due to prelamin A accumulation (Slide 24 [5]).
HD children’s cells have upregulated DNA damage markers and increased osteogenic signalling [11]. Their vessels calcified more readily than the cells from control children, since DNA damage was driving the calcification and the osteogenic differentiation in these cells. The calcifications, as well as the inflammatory markers are downregulated by ATM signalling inhibition. The children on HD had high levels of SASP factors BMP2, IL6, and osteoprotegerin in their circulation, suggesting that old arteries were releasing the SASP factors into the circulation and driving tissue damage not only in the vasculature but also in the remote tissues. When children on HD had their coronary artery calcification measured by CT scan, it was shown that the non-calcified group had lower levels of SASP factors in their circulation, as compared to the HD children with calcified arteries, suggesting again a correlation between senescence-associative inflammation and calcification (Slides 26-28 [5]).
Additionally, PARP DNA damage signalling pathway produces the molecule PAR, which forms the first nidus of mineralization. PAR polymerase (PARP) inhibitors can block calcification, as they successfully blocked the calcification in vivo in the rat model of CKD [12].


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