Objective—: Prevalence of vitamin D (VD) deficiency and its association with the risk of cardiovascular disease prompted us to evaluate the effect of VD status on lipid metabolism and atherosclerosis in hypercholesterolemic microswine. Approach and Results—: Yucatan microswine were fed with VD-deficient (0 IU/d), VD-sufficient (1000 IU/d), or VD-supplemented (3000 IU/d) high-cholesterol diet for 48 weeks. Serum lipids and 25(OH)-cholecalciferol levels were measured biweekly. Histology and biochemical parameters of liver and arteries were analyzed. Effect of 1,25(OH) ₂ D ₃ on cholesterol metabolism was examined in human hepatocyte carcinoma cell line (HepG2) and human monocytic cell line (THP-1) macrophage-derived foam cells. VD deficiency decreased plasma high-density lipoprotein levels, expression of liver X receptors, ATP-binding membrane cassette transporter A1, and ATP-binding membrane cassette transporter G1 and promoted cholesterol accumulation and atherosclerosis in hypercholesterolemic microswine. VD promoted nascent high-density lipoprotein formation in HepG2 cells via ATP-binding membrane cassette transporter A1–mediated cholesterol efflux. Cytochrome P450 (CYP)27B1 and VD receptor were predominantly present in the CD206 ⁺ M2 macrophage foam cell–accumulated cores in coronary artery plaques. 1,25(OH) ₂ D ₃ increased the expression of liver X receptors, ATP-binding membrane cassette transporter A1, and ATP-binding membrane cassette transporter G1 and promoted cholesterol efflux in THP-1 macrophage–derived foam cells. 1,25(OH) ₂ D ₃ decreased intracellular free cholesterol and polarized macrophages to M2 phenotype with decreased expression of tumor necrosis factor-α, interleukin-1β, interleukin-6 under lipopolysaccharide stimulation. 1,25(OH) ₂ D ₃ markedly induced CYP27A1 expression via a VD receptor–dependent c-Jun N-terminal kinase (JNK) 1/2 signaling pathway and increased 27-hydroxycholesterol levels, which induced liver X receptors, ATP-binding membrane cassette transporter A1, and ATP-binding membrane cassette transporter G1 expression and stimulated cholesterol efflux that was inhibited by VD receptor antagonist and JNK1/2 signaling inhibitor in THP-1 macrophage–derived foam cell. Conclusions—: VD protects against atherosclerosis in hypercholesterolemic swine via controlling cholesterol efflux and macrophage polarization via increased CYP27A1 activation.

Inflammation and extracellular matrix (ECM) disorganization following the rotator cuff tendon injuries (RCTI) delay the repair and healing process and the molecular mechanisms underlying RCTI pathology are largely unknown. Here, we examined the role of HMGB1 and NLRP3 inflammasome pathway in the inflammation and ECM disorganization in RCTI. This hypothesis was tested in a tenotomy-RCTI rat model by transecting the RC tendon from the humerus. H&E and pentachrome staining revealed significant changes in the morphology, architecture and ECM organization in RC tendon tissues following RCTI when compared with contralateral control. Severity of the injury was high in the first two weeks with improvement in 3–4 weeks following RCTI, and this correlated with the healing response. The expression of proteins associated with increased HMGB-1 and upregulation of NLRP3 inflammasome pathway, TLR4, TLR2, TREM-1, RAGE, ASC, Caspase-1, and IL-1β, in the first two weeks following RCTI followed by decline in 3–4 weeks. These results suggest the association of inflammatory responses and ECM disorganization with HMGB1 upregulation and NLRP3 inflammasome activation in the RC tendons and could provide novel target(s) for development of better therapeutic strategies in the management of RCTI.

Objective—: The role of vitamin D deficiency in coronary artery disease (CAD) progression is uncertain. Chronic inflammation in epicardial adipose tissue (EAT) has been implicated in the pathogenesis of CAD. However, the molecular mechanism underlying vitamin D deficiency–enhanced inflammation in the EAT of diseased coronary arteries remains unknown. We examined a mechanistic link between 1,25-dihydroxyvitamin D–mediated suppression of nuclear factor-κB (NF-κB) transporter, karyopherin α4 (KPNA4) expression and NF-κB activation in preadipocytes. Furthermore, we determined whether vitamin D deficiency accelerates CAD progression by increasing KPNA4 and nuclear NF-κB levels in EAT. Approach and Results—: Nuclear protein levels were detected by immunofluorescence and Western blot. Exogenous KPNA4 was transported into cells by a transfection approach and constituted lentiviral vector. Swine were administered vitamin D–deficient or vitamin D–sufficient hypercholesterolemic diet. After 1 year, the histopathology of coronary arteries and nuclear protein expression of EAT were assessed. 1,25-dihydroxyvitamin D inhibited NF-κB activation and reduced KPNA4 levels through increased vitamin D receptor expression. Exogenous KPNA4 rescued 1,25-dihydroxyvitamin D–dependent suppression of NF-κB nuclear translocation and activation. Vitamin D deficiency caused extensive CAD progression and advanced atherosclerotic plaques, which are linked to increased KPNA4 and nuclear NF-κB levels in the EAT. Conclusions—: 1,25-dihydroxyvitamin D attenuates NF-κB activation by targeting KPNA4. Vitamin D deficiency accelerates CAD progression at least, in part, through enhanced chronic inflammation of EAT by upregulation of KPNA4, which enhances NF-κB activation. These novel findings provide mechanistic evidence that vitamin D supplementation could be beneficial for the prevention and treatment of CAD.

Mitochondrial function following rotator cuff tendon injury (RCI) influences the tendon healing. We examined the mitochondrial morphology and function under hypoxia in the shoulder tendon tissue from surgically-induced tenotomy-RCI rat model and cultured swine tenocytes. The tendon tissue was collected post-injury on 3–5 (Group-A), 10–12 (Group-B), and 22–24 (Group-C), days and the corresponding contralateral tendons were used as control for each group. There was higher protein expression of citrate synthase (P < 0.0001) [10.22 MFI (mean fluorescent intensity)] and complex-1 (P = 0.0008) (7.86 MFI) in Group-A and Group-B that decreased in Group-C [(P = 0.0201) (5.78 MFI and (P = 0.7915) (2.32 MFI), respectively] compared to control tendons. The ratio of BAX:Bcl2 (Bcl2 associated x protein:B cell lymphoma 2) in RCI tendons increased by 50.5% (Group-A) and 68.4% (Group-B) and decreased by 25.8% (Group-C) compared to normoxic controls. Hypoxia increased β-tubulin expression (P = 0067) and reduced PGC1-α (P = 0412) expression in the isolated swine tenocytes with no effect on the protein expression of Complex-1 (P = 7409) and citrate synthase (P = 0.3290). Also, the hypoxic tenocytes exhibited about 4-fold increase in mitochondrial superoxide (P < 0.0001), altered morphology and mitochondrial pore integrity, and increase in mitochondrial density compared to normoxic controls. These findings suggest the critical role of mitochondria in the RCI healing response.

Osteoarthritis (OA) of the knee joint is a degenerative process resulting in cartilage loss. Recent evidence suggests that OA is not merely a disease of cartilage but a disease of the entire knee joint and that inflammation may play an important role. OA has been associated with vitamin D deficiency. Vitamin D as an immunomodulator and anti-inflammatory agent may attenuate inflammation in the knee. The aim of this study was to assess the anti-inflammatory effect of vitamin D on inflammation in the knee. This study was conducted with 13 microswine on a high cholesterol diet categorized into three groups of vitamin D-deficient, vitamin D-sufficient, and vitamin D supplementation. After 1 year, microswine were killed, and their knee joint tissues were harvested. Histological and immunofluorescence studies were carried out on the tissue specimens to evaluate the effect of vitamin D status. Histological and immunofluorescence studies of the knee joint tissues showed (1) increased inflammation in the knee joint tissues, (2) fatty infiltration in quadriceps muscle, patellar tendon, and collateral ligaments, and (3) chondrocyte clustering in the vitamin D-deficient and vitamin D-sufficient groups compared with the vitamin D supplementation group. Architectural distortion of the quadriceps muscle, patellar tendon, and collateral ligaments was also seen in the areas of inflammatory foci and fatty infiltration in the vitamin D-deficient group. Decreased inflammation and fatty infiltration in the vitamin D supplementation group suggest the potential role of vitamin D in attenuating inflammation and fatty infiltration as well as in protecting the architecture of the tissue in the knee joint.