Medial vascular calcification (MVC) is a highly prevalent disease associated with a high risk of severe, potentially lethal, complications. While animal studies may not systematically be circumvented, in vitro systems have been proven useful to study disease physiopathology. In the context of MVC, the absence of a clinically relevant standardized in vitro method prevents the appropriate comparison and overall interpretation of results originating from different experiments. The aim of our study is to establish in vitro models mimicking in vivo vascular calcification and to select the best methods to unravel the mechanisms involved in MVC. Human aortic smooth muscle cells and rat aortic rings were cultured in different conditions. The influence of fetal calf serum (FCS), alkaline phosphatase, phosphate and calcium concentrations in the medium were evaluated. We identified culture conditions, including the herein reported Aorta Calcifying Medium (ACM), which allowed a reproducible and specific medial calcification of aortic explants. Studying cells and aortic explants cultured, the involvement of bone morphogenetic protein 2 (BMP2) pathway, fibrosis and apoptosis processes in in vitro MVC were demonstrated. Expression of osteoblastic markers was also observed suggesting the occurrence of transdifferentiation of smooth muscle cells to osteoblasts in our models. The use of these models will help researchers in the field of vascular calcification to achieve reproducible results and allow result comparison in a more consistent way.

Increased senescent cell burden and dysregulation of the nuclear factor erythroid 2–related factor 2 (NRF2) pathway have been associated with numerous age-related pathologies; however, their role in promoting vascular calcification (VC) in chronic kidney disease (CKD) has yet to be determined. We investigated whether senescence and NRF2 pathways may serve as drivers of uremia-induced VC using three complementary approaches: a novel model of induced VC in 5/6-nephrectomized rats supplemented with high phosphate and vitamin D; epigastric arteries from CKD patients with established medial calcification; and vascular smooth muscle cells (VSMCs) incubated with uremic serum. Expression of p16Ink4a and p21Cip1, as well as γ-H2A-positive cells, confirmed increased senescent cell burden at the site of calcium deposits in aortic sections in rats, and was similarly observed in calcified epigastric arteries from CKD patients through increased p16Ink4a expression. However, uremic serum-induced VSMC calcification was not accompanied by senescence. Expression of NRF2 and downstream genes, Nqo1 and Sod1, was associated with calcification in uremic rats, while no difference was observed between calcified and non-calcified EAs. Conversely, in vitro uremic serum-driven VC was associated with depleted NRF2 expression. Together, our data strengthen the importance of senescence and NRF2 pathways as potential therapeutic options to combat VC in CKD.

Excessive fat consumption leads to the development of ectopic adipose tissues, affecting the organs they surround. Peripancreatic adipose tissue is implicated in glucose homeostasis regulation and can be impaired in obesity. High palm oil consumption’s effects on health are still debated. We hypothesised that crude and refined palm oil high-fat feeding may have contrasting effects on peripancreatic adipocyte hypertrophy, inflammation and lipid oxidation compound production in obese rats. In Wistar rats, morphological changes, inflammation and isoprostanoid production following oxidative stress were assessed in peripancreatic adipose tissue after 12 weeks of diets enriched in crude or refined palm oil or lard (56% energy from fat in each case) versus a standard chow diet (11% energy from fat). Epididymal white and periaortic brown adipose tissues were also included in the study. A refined palm oil diet disturbed glucose homeostasis and promoted lipid deposition in periaortic locations, as well as adipocyte hypertrophy, macrophage infiltration and isoprostanoid (5-F2c-isoprostane and 7(RS)-ST-Δ8-11-dihomo-isofuran) production in peripancreatic adipose tissue. Crude palm oil induced a lower impact on adipose deposits than its refined form and lard. Our results show that the antioxidant composition of crude palm oil may have a protective effect on ectopic adipose tissues under the condition of excessive fat intake.

Vascular calcification is a risk factor for cardiovascular and kidney diseases. Medial calcification may differently affect the arterial tree depending on vessel location and smooth muscle injury. The aim was to map the anatomical distribution of vascular calcifications on different arteries and artery locations, in cultured artery rings (ex vivo) and in a rat model of elastocalcinosis (in vivo). Vascular calcification was assessed histologically (von Kossa staining of the media) and by calcium content measurement. Arteries of different sizes were harvested from untreated rats for ring culture and from the vitamin D3-nicotine (VDN) rat model for direct observation. When cultured in pro-calcifying conditions, thoracic aorta exhibited similar calcification from the arch to the diaphragm. Calcification increased in abdominal aorta along with the reduction in cross sectional area. Carotid and renal arteries exhibited similar ex vivo calcification. In VDN rats, calcification was greater in carotid artery than in aorta, and was accompanied by fibrosis and apoptosis. Ex vivo, calcification was increased by the induction of lesions on arteries. Along the vascular tree, calcification of the arterial wall increases with the narrowing of vessels in ex vivo ring culture and in vivo. The observed differences represent local susceptibility of the vessels to the calcifying processes.

Background and Aims Vascular calcification (VC) is a consequence of ageing that confers development of future cardiovascular events. Accumulation of senescent cells can lead to structural and functional abnormalities in vessel wall towards increased stiffness, reduced compliance and impaired contractile and dilatory capacity. Thus, accumulation of senescent cells in the arterial wall could also contribute to the pathophysiology of VC. To test this hypothesis, the presence of cellular markers of senescence, p16, p21 and NRF2, was assessed in aorta from rat model of VC associated with chronic kidney disease (CKD). Method Six-week-old Sprague-Dawley rats underwent 5/6th subtotal nephrectomy (SNx, n=6) or no surgery (Control, n=1). After 8 weeks of renal failure, the regular chow was supplemented with high phosphate (1.2%) and vitamin D (1 µg/day, 5 days per week) for 1 or 4 weeks to initiate vascular calcification (SNx-VC group). At the end of the protocol (Figure 1), blood pressure was measured, and thoracic aorta was taken for determination of calcification by Von Kossa staining, and protein and gene expressions of p16, p21, and NRF2 by immunostaining and qPCR, respectively. Results After 4 weeks of dietary intervention, SNx rats showed an increase in pulse pressure (88±15mmHg vs 35 mmHg for the control). Marked VC was also observed in these animals, 17% of calcified area vs <1 % in the control rat. Calcification was focal giving strong and no calcified areas on the same aorta. Expressions of p16, p21and NRF2 proteins were enhanced at the site of calcification in the SNx-VC 4-week (4W) group whereas it was unchanged in aortic media without calcification compared to the control rat (Figure 2). In SNx-VC 1-week (1W) rats, no change in pulse pressure or vascular calcification was observed without obvious changes for staining patterns of selected markers. Using qPCR we found p16 gene expression to be higher in the most calcified aortas (4W) (fold-change = 4.32 vs 1W) and p21 gene expression to be slightly increased (fold-change = 0.53 vs 1W). NRF2 gene expression was enhanced in 1W group compared to control, but decreased in the most calcified samples (4W). Conclusion This pilot study suggests that uraemia-induced cellular senescence accompanies VCs, as suggested by the modified expression of p16 or NRF2 genes. Our observations deserve exploration in larger studies using additional senescence markers for validation. If so, cellular senescence kinetics will be evaluated in order to test whether senolytics compounds could be a therapeutic option to arrest VC in CKD.