Warfarin treatment of apoE deficient mice on Western Type Diet does not influence plaque size but increases vascular calcification.

<p>ApoE^−/− mice developed atherosclerotic lesions in the aortic arch and carotid arteries when maintained on Western type diet (WTD) for 12 weeks (A). From baseline mice were fed with WTD plus vitamin K (VK₁) or WTD plus vitamin K and warfarin (VK₁&W) for the duration of one week or four weeks. Growth of intimal area was not significantly affected by warfarin. Vascular calcium was determined by AAS and revealed significant increase in calcium at 1 and 4 weeks warfarin treatment. Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05.</p

Baseline characteristics of patients not on VKA treatment.

<p>Continuous variables are presented as means ± SD, while categorical variables are expressed as number (percentage). BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein.</p

Duration of VKA treatment significantly increases the amount of coronary calcification and number of calcified stenotic plaques.

<p>A non-enhanced scan was performed to measure the Coronary artery calcification (Agatston score). Panel A shows a significant increase in Agatston score in VKA users (p = 0.029), as patients use VKA for a longer time (T = tertile of VKA use). This increase is not visible in individually matched patients not on VKA (panel B; p = 0.965). Next, CT-angiography was performed. All coronary segments were assessed for plaque presence and plaque morphology. In patients using VKA, a significant trend (p = 0.009) was seen towards a higher percentage of calcified plaques in those patients treated longest with VKA (panel C). In contrast, this was not the case for the non-VKA users (panel D). Coronary plaques were identified and categorized as calcified (closed circles), mixed calcified (diamonds) and non-calcified (open circles). T1, T2 and T3 indicate 1^st, 2^nd and 3^rd tertile respectively.</p

Warfarin treatment of apoE^−/− mice increases apoptosis in atherosclerotic plaque.

<p>Vascular calcification is closely linked to apoptosis. Therefore we stained sections for caspase-3. Moreover, all animals were injected with annexin A5-biotin, a protein with high affinity for phosphatidylserine (PS). Both cleaved caspase-3 (A,C) and annexin A5-biotin (B,D) increased in the warfarin treatment, with annexin A5 accumulation significantly increased after 4 weeks of warfarin treatment (B). Annexin A5 staining using AP vector blue confirmed PS externalization at different sites in the atherosclerotic plaques. Activated and apoptotic macrophages are known to express PS on their surface. We found co-localization of macrophages in the shoulder of the atherosclerotic plaque (Mac3; F) and PS (annexin A5 stained with AP vector red; E), indicating activated and apoptotic macrophages. In the inset annexin A5 (blue) localizes with Mac-3 staining (red/brown), indicating activated macrophages. Moreover, at baseline annexin A5 was only incidentally present at the surface of the plaque (G), whereas at the 4 weeks warfarin treatment, annexin A5 stained significantly positive (H). Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05. i, intima; m, media; l, lumen.</p

Warfarin affects carboxylation of MGP in atherosclerotic plaque.

<p>To confirm action of warfarin locally in the plaque we stained plaques for ucMGP and cMGP. UcMGP is the result of vitamin K-deficiency. Within one week of warfarin treatment the amount of cMGP (A,E) decreased significantly and decreased even further after 4 weeks of warfarin treatment. The decrease in cMGP was accompanied by an increase in ucMGP (B,F). Immunohistochemistry for cMGP and ucMGP (E,F) showed increased amounts of ucMGP compared with cMGP in apoE^−/− mice on warfarin, whereas apoE^−/− mice on control diet had predominantly cMGP (C,D). cMGP indicates carboxylated MGP; ucMGP, uncarboxylated MGP. Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05, **P<0.01. i, intima; m, media; l, lumen.</p

Weight and blood characteristics of apoE^−/− mice at baseline, and after one or four weeks of treatment.

<p>WTD, Western type diet.</p

Warfarin treatment rapidly increases medial and intimal plaque calcification in apoE<i>^−/−</i> mice.

<p>ApoE−/− mice received WTD for three month (baseline) and subsequently control diet (WTD plus VK₁) or warfarin (WTD plus VK₁&W). Von Kossa stained calcified plaques were scored for medial (A,C) and intimal plaque calcification (B,D). In addition calcification was categorized as microcalcification (E, arrow heads) and macrocalcification (F, arrows). Microcalcifications occur either alone or in conjunction with macrocalcification. Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05, **P<0.01, ***P<0.001. i, intima; m, media; l, lumen; a, adventitia.</p

mRNA expression levels of aortic tissue (n = 3) of calcification biomarkers at control and warfarin treatment at 4 weeks time point.

<p>mRNA expression levels of aortic tissue (n = 3) of calcification biomarkers at control and warfarin treatment at 4 weeks time point.</p

Warfarin induces medial VSMC loss and increases outward remodeling of atherosclerotic plaques, both indications of an instable plaque phenotype.

<p>An unexpected feature of warfarin treatment was the significantly increased number of outward remodeled plaques (B,D). Outward remodeling is, like plaque calcification, a feature of the instable rupture-prone atherosclerotic plaque. To explain this phenotype induced by warfarin we stained sections for the VSMC marker αSMactin. We found that warfarin treatment was associated with a significant loss of medial VSMCs (A,C). Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05, **P<0.01. i, intima; m, media; l, lumen; o, outward remodeled plaque.</p

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

Background Sparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis. Methods PROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850. Findings Between Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals. Interpretation Over 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function.</p