Background: No pharmacological treatment exists to prevent or stop the calcification process of aortic valves causing aortic stenosis. The aim of this study was to develop a robust model of induced calcification in whole aortic valve leaflets which could be suitable for studies of the basic mechanisms and for testing potentially inhibitory drugs.Methods: Pig hearts were obtained from a commercial abattoir. The aortic valve leaflets were dissected free and randomized between experimental groups. Whole leaflets were cultured in individual wells. Two growth media were used for cultivation: standard growth medium and an antimyofibroblastic growth medium. The latter was employed to inhibit contraction of the leaflet into a ball-like structure. Calcification was induced in the growth medium by supplementation with an osteogenic medium. Leaflets were cultivated for four weeks and medium was changed every third day. To block calcification, the inhibitor SNF472 (a formulation of the hexasodium salt of myo-inositol hexaphosphate hexasodium salt) was used at concentrations between 1 and 100 µM. After cultivation for four weeks the leaflets were snap frozen in liquid nitrogen and kept at −80 °C until blind assessment of the calcium amount in leaflets by inductively coupled plasma optical emission spectroscopy. For statistical analysis, a Kruskal–Wallis test with Dunn’s post-test was applied.Results: Osteodifferentiation with calcium accumulation was in principle absent when standard medium was used. However, when the antimyofibroblastic medium was used, a strong calcium accumulation was induced (p = 0.006 compared to controls), and this was blocked in a dose-dependent manner by the calcification inhibitor SNF472 (p = 0.008), with an EC50 of 3.3 µM.Conclusion: A model of experimentally induced calcification in cultured whole leaflets from porcine aortic valves was developed. This model can be useful for studying the basic mechanisms of valve calcification and to test pharmacological approaches to inhibit calcification.

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Background and Aims Peripheral artery disease (PAD) is a common co-morbidity in end-stage kidney disease (ESKD) patients undergoing dialysis. The prominent vascular calcification in these patients is associated with severity of symptoms and adverse outcomes. Although there are medical therapies approved for PAD, none of them has been specifically approved por PAD-ESKD. For example, cilostazol is approved for PAD but its use is limited in PAD-ESKD patients. Surgical interventions are challenging in these patients due to their specific vascular calcification and show worse outcomes. Therefore, the aims of this study were to evaluate the effects of the investigational drug SNF472, a selective inhibitor of vascular calcification, on blood perfusion and limb functional recovery in a Vitamin D3 (VitD)-induced rat model of arterial calcification and limb ischemia. Method Three consecutives daily subcutaneous (s.c) doses of VitD were administered to 66 male Sprague Dawley rats to induce limb ischemia. Rats were randomized into four groups at day 5 after the start of VitD treatment (when all parameters were measured in a satellite group) and were treated for nine days with placebo s.c., placebo peroral (p.o.), SNF472 (40 mg/kg/day, s.c.) or cilostazol (40 mg/kg/day, p.o.). An additional control group did not receive VitD (sham) and was administered with placebo s.c. during these last nine days. Posterior limb blood perfusion was measured using laser Doppler imaging at baseline (before calcification induction), day 4 (before treatment start) and day 13 (end of treatment). Walking ability was evaluated by measuring Maximum Walking Distance (MWD) and Maximum Walking Time (MWT) using a rat treadmill at baseline, day 4 and day 11. Rats were sacrificed at day 13, and heart and femoral arteries were collected for calcium analysis. Results Administration of VitD induced heart and femoral artery calcification by day 5. This calcification was associated with decreased limb blood perfusion and impairment of walking ability (both MWT and MWD) compared to sham. Treatment with SNF472 inhibited calcification progression in femoral arteries by 41% and in heart by 56% compared to placebo. The inhibition of calcification progression by SNF472 led to a 29% increase in limb blood perfusion (p< 0.001) and a significant improvement in walking ability (49% in MWD and 43% in MWT; p< 0.05) compared to placebo. Calcification inhibition in femoral arteries was positively correlated with both MWD and MWT (p< 0.0001). No effects of cilostazol were observed in tissue calcification, limb blood perfusion or walking ability. Conclusion SNF472 attenuates the progression of vascular calcification and improves blood perfusion and the functional parameters MWT and MWD in a rat model of PAD vascular calcification. These results support investigation of SNF472 as a potential therapy for PAD-ESKD patients who have vascular dysfunction due to a high degree of arterial calcification.

Objectives: Venous ulcers (VUs) are well-studied in high-income settings and are known to have a significant impact on patients' overall quality of life. However, VUs are generally considered a disease of wealthier countries and have not received the attention they deserve in low-and middle-income countries (LMICs). This study aimed to explore the scope and accessibility of published data on venous ulcers in LMICs.Methods: We conducted a scoping literature search using PubMed, Medline, Embase, and CINAHL. Included studies were peer-reviewed, reported data on VUs, and were performed in LMICs as specified by the 2020 World Bank classifications. Studies performed in high-income countries (gross national income [GNI] per capita greater than $12,535 annually), systematic/scoping/literature reviews, and editorials were excluded. There were no language or date restrictions to our search.Results: In total, 2831 studies were found, of which 312 were included in the final analysis. Most studies were in English (61%), with 22% in Portuguese and 10% in Chinese. All studies included patients with C5 or C6 chronic venous disease, and over 70% of studies were conducted in a tertiary care hospital. Over one-third (38%) focused on treatment for VUs (38% on wound care, 13% on surgical treatment, 8% on ablation procedures). In total, 28 countries were represented. The overwhelming majority of studies were conducted in Brazil (56%) and China (14%), both of which are classified as upper-middle income countries (GNI per capita between $4,046 and $12,535 annually) and have a surgical/obstetric/anesthesia provider density higher than the recommended 20 per 100,000 population. Only 2 studies were performed in low-income countries (GNI per capita below $1,035 annually), both of which reported significant burden due to venous disease among their studied populations. Though all of the regions defined by the World Health Organization were represented, only 10.6% of LMICs in the African region were represented, compared with over 40% of the LMICs in the Pan-American region (Fig).Conclusions: VUs affect patients in low, low-middle, and upper-middle income settings. However, more studies are needed to truly quantify this burden, particularly at the district and community levels. Furthermore, much more work is needed to document the deleterious impact of VUs in African regions. Increased vascular surgical capacity-building and outreach in LMICs, both through intraregional and international partnerships, are essential to understand the burden of VUs in these regions and to scale up local vascular provider density and access to treatment for the patients affected.

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