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Recent studies have found an association between high volumes of physical activity and increased levels of coronary artery calcification (CAC) among older male endurance athletes, yet the underlying mechanisms have remained largely elusive. Potential mechanisms include greater exposure to inflammatory cytokines, reactive oxygen species and oxidised low-density lipoproteins, as acute strenuous physical activity has been found to enhance their systemic release. Other possibilities include post-exercise elevations in circulating parathyroid hormone, which can modify the amount and morphology of calcific plaque, and long-term exposure to non-laminar blood flow within the coronary arteries during vigorous physical activity, particularly in individuals with pre-existing atherosclerosis. Further, although the association has only been identified in men, the role of testosterone in this process remains unclear. This brief review discusses the association between high-volume endurance exercise and CAC in older men, elaborates on the potential mechanisms underlying the increased calcification, and provides clinical implications and recommendations for those at risk.

In calcific aortic valve disease, the valve cusps undergo retraction, stiffening, and nodular calcification. The inflammatory cytokine, tumor necrosis factor (TNF)-α, contributes to valve disease progression; however, the mechanisms of its actions on cusp retraction and stiffening are unclear. We investigated effects of TNF-α on murine aortic valvular interstitial cells (VICs) within three-dimensional, free-floating, compliant, collagen hydrogels, simulating their natural substrate and biomechanics. TNF-α increased retraction (percentage of diameter), stiffness, and formation of macroscopic, nodular structures with calcification in the VIC-laden hydrogels. The effects of TNF-α were attenuated by blebbistatin inhibition of myosin II–mediated cytoskeletal contraction. Inhibition of actin polymerization with cytochalasin-D, but not inhibition of Rho kinase with Y27632, blocked TNF-α–induced retraction in three-dimensional VIC hydrogels, suggesting that actin stress fibers mediate TNF-α–induced effects. In the hydrogels, inhibitors of NF-κB blocked TNF-α–induced retraction, whereas simultaneous inhibition of c-Jun N-terminal kinase was required to block TNF-α–induced stiffness. TNF-α also significantly increased collagen deposition, as visualized by Masson's trichrome staining, and up-regulated mRNA expression of discoidin domain receptor tyrosine kinase 2, fibronectin, and α-smooth muscle actin. In human aortic valves, calcified cusps were stiffer and had more collagen deposition than noncalcified cusps. These findings suggest that inflammation, through stimulation of cytoskeletal contractile activity, may be responsible for valvular cusp retraction, stiffening, and formation of calcified nodules.

ABSTRACTBackgroundAlthough physician-scientists generally contribute to the scientific enterprise by providing a breadth of knowledge complementary to that of other scientists, it is a challenge to recruit, train, and retain physicians in a research career pathway.Objective To assess the outcomes of a novel program that combines graduate coursework and research training with subspecialty fellowship.Methods A retrospective analysis was conducted of career outcomes for 123 physicians who graduated from the program during its first 20 years (1993–2013). Using curricula vitae, direct contact, and online confirmation, data were compiled on physicians' subsequent activities and careers as of 2013. Study outcomes included employment in academic and nonacademic research, academic clinical or private practice positions, and research grant funding.Results More than 80 of graduates were actively conducting research in academic, institutional, or industrial careers. The majority of graduates (71) had academic appointments; a few (20) were in private practice. Fifty percent had received career development awards, and 19 had received investigator-initiated National Institutes of Health (NIH) R01 or equivalent grants. Individuals who obtained a PhD during subspecialty training were significantly more likely to have major grant funding (NIH R series or equivalent) than those who obtained a Master of Science in Clinical Research. Trainees who obtained a PhD in a health services or health policy field were significantly more likely to have research appointments than those in basic science.Conclusions Incorporation of graduate degree research, at the level of specialty or subspecialty clinical training, is a promising approach to training and retaining physician-scientists.

Background:Endothelial-to-mesenchymal transition (EndMT), the process whereby endothelial cells (ECs) transition into multipotent stem cell-like cells, has been implicated in a variety of cardiovascular pathologies, including cardiovascular calcification. EndMT is a dynamic mechanical process that involves detachment of transitioning ECs from adjacent cells and active migration of these cells from the endothelium into the interstitium. While gene and protein expression profiles of ECs undergoing EndMT have been well characterized, less is known about the mechanical properties, or “mechanotype,” of ECs as they undergo EndMT.Methods/Results:Human aortic endothelial cells (HAEC) were treated with the inflammatory cytokine, tumor necrosis factor-α (TNF-α), to induce inflammatory EndMT. Real-time PCR analyses showed that TNF-α significantly reduced the expression of endothelial markers (PECAM1, NOS3) by 24 hours and significantly increased the expression of mesenchymal markers (COL1A1, COL3A1, FN1, CNN1) starting at 48 hours of treatment. Mechanotyping of EC was performed by evaluating cell deformability with the parallel microfiltration (PMF) technique. PMF is a novel method that determines relative deformability by filtering cells across a porous membrane. Results showed that TNF-α has a biphasic effect on EC deformability, increasing the deformability after 24 hours, while decreasing deformability after 48 hours of treatment. Deformability decreased further with 96 hours of TNF-α treatment. Transwell migration assays revealed that TNF-α increased migration of ECs through a porous membrane, whereas EC migration was not enhanced when assessed by a scratch wound assay.Conclusion:Our results demonstrate that ECs undergo dynamic changes in their mechanotype during inflammatory EndMT. A deeper understanding of the mechanisms governing these changes may help inform therapeutic targets for EndMT-associated disease processes.

The reaction and diffusion of morphogens is a mechanism widely used to explain many spatial patterns in physics, chemistry and developmental biology. However, because experimental control is limited in most biological systems, it is often unclear what mechanisms account for the biological patterns that arise. Here, we study a biological model of cultured vascular mesenchymal cells (VMCs), which normally self-organize into aggregates that form into labyrinthine configurations. We use an experimental control and a mathematical model that includes reacting and diffusing morphogens and a third variable reflecting local cell density. With direct measurements showing that cell motility was increased ninefold and threefold by inhibiting either Rho kinase or non-muscle myosin-II, respectively, our experimental results and mathematical modelling demonstrate that increased motility alters the multicellular pattern of the VMC cultures, from labyrinthine to a pattern of periodic holes. These results suggest implications for the tissue engineering of functional replacements for trabecular or spongy tissue such as endocardium and bone.

Vascular calcification is associated with an increased risk of cardiovascular and all-cause mortality. In the past decade, the prevalence, significance, and regulatory mechanisms of vascular calcification have gained increasing recognition. In this Review, Drs. Sage, Tintut and Demer discuss our knowledge of the regulatory mechanisms for both atherosclerotic and medial calcification.

Cholesterol is an important molecule that plays a key role in regulating cellular differentiation and function. Although the possible role of lipids has been implicated in regulating osteoblastic cells, the role of cholesterol in that process is not well defined. In this study we have examined the role of the cellular cholesterol biosynthetic pathway on osteoblastic differentiation of marrow stromal cells (MSCs). Treatment of pluripotent mouse MSCs M2–10B4 with inhibitors of the cholesterol biosynthetic pathway mevastatin or mevinolin inhibited the maturation of these cells into functional osteoblastic cells. This was determined by the inhibition of the activity and expression of alkaline phosphatase (ALP), a key enzyme involved in differentiation and mineralization of osteoblastic cell cultures, as well as inhibition of mineralization. Mevastatin treatment did not affect expression of the osteoblast‐specific gene osteocalcin (OCN). Furthermore, promoter‐reporter studies in MSCs showed that mevastatin inhibited activity of the ALP gene promoter, suggesting regulation by derivatives of the cholesterol biosynthetic pathway. The effects of mevastatin and mevinolin were reversed by mevalonate but not by geranylgeraniol or farnesol, intermediates in the cholesterol biosynthetic pathway. Altogether, these results suggest that products of the cholesterol biosynthetic pathway are important for proper development of MSCs into functional osteoblastic cells capable of forming a mineralized matrix. Identification of those molecules may provide new therapeutic approaches to prevent the decline in osteoblastic activity in osteoporosis and aging.

Cholesterol is an important molecule that plays a key role in regulating cellular differentiation and function. Although the possible role of lipids has been implicated in regulating osteoblastic cells, the role of cholesterol in that process is not well defined. In this study we have examined the role of the cellular cholesterol biosynthetic pathway on osteoblastic differentiation of marrow stromal cells (MSCs). Treatment of pluripotent mouse MSCs M2–10B4 with inhibitors of the cholesterol biosynthetic pathway mevastatin or mevinolin inhibited the maturation of these cells into functional osteoblastic cells. This was determined by the inhibition of the activity and expression of alkaline phosphatase (ALP), a key enzyme involved in differentiation and mineralization of osteoblastic cell cultures, as well as inhibition of mineralization. Mevastatin treatment did not affect expression of the osteoblast‐specific gene osteocalcin (OCN). Furthermore, promoter‐reporter studies in MSCs showed that mevastatin inhibited activity of the ALP gene promoter, suggesting regulation by derivatives of the cholesterol biosynthetic pathway. The effects of mevastatin and mevinolin were reversed by mevalonate but not by geranylgeraniol or farnesol, intermediates in the cholesterol biosynthetic pathway. Altogether, these results suggest that products of the cholesterol biosynthetic pathway are important for proper development of MSCs into functional osteoblastic cells capable of forming a mineralized matrix. Identification of those molecules may provide new therapeutic approaches to prevent the decline in osteoblastic activity in osteoporosis and aging.

Vascular calcification occurs more often and earlier in patients with end-stage renal disease than in normal controls. It is a regulated biological process following many of the cellular and molecular programs in osteogenesis. This review summarizes some of the regulatory mechanisms that may explain its severity in renal patients.

Lipid oxidation products promote atherosclerosis and may also affect osteoporosis. We showed previously that oxidized lipids including 8-isoprostaglandin E2 (isoPGE2) inhibit osteoblastic differentiation of preosteoblasts. Since osteoporosis is mediated both by decreased osteoblastic bone formation and by increased osteoclastic bone resorption, we assessed whether oxidized lipids regulate the osteoclastic potential of marrow hematopoietic cells. Treatment of marrow-derived preosteoclasts with isoPGE2 enhanced osteoclastic differentiation as evidenced by increased tartrate-resistant acid phosphatase (TRAP) activity and multinucleation, which were inhibited by calcitonin, and increased numbers of resorption pits. The enhanced osteoclastic differentiation by isoPGE2 was observed whether preosteoclasts were in coculture with stromal cells or in monoculture in the presence of receptor-activated NFκB ligand (RANKL) and macrophage colony-stimulating factor. Receptor antagonist studies suggest that isoPGE2 effects were mediated by prostaglandin receptor subtypes EP2/DP on preosteoclasts and subtype EP1 and thromboxane receptors on stromal/osteoblast cells. The enhanced TRAP activity was also inhibited by cAMP-dependent protein kinase inhibitors, and isoPGE2 elevated intracellular cAMP levels of preosteoclast monocultures. Other oxidized lipids also enhanced the TRAP activity of preosteoclast monocultures. These data suggest that isoPGE2 enhances osteoclastic differentiation of marrow preosteoclasts and that this regulation occurs via the cAMP-dependent protein kinase pathway.

Calcification presents important clinical implications in cardiovascular diseases, especially in coronary arteries. Epidemiological evidence has shown the coexistence of vascular calcification with both atherosclerosis and osteoporosis, and increasing evidence has shown the role of hyperlipidemia and atherogenic phospholipids in vascular calcification. The etiology of vascular calcification is also increasingly recognized as an active process. Vascular calcification initiates with matrix vesicle formation and mineralization following a process similar to that in bone. In addition, many bone regulatory factors have been shown to be present in calcified atherosclerotic lesions. In this review, we focus on the new developments emerging during the past year in regulation of vascular calcification. Regulatory factors include matrix GLA protein, the phosphate cotransporter Pit-1, a calcium-sensing receptor related factor, osteoprotegerin, leptin, bisphosphonates and oxidized lipids. Some of these, including oxidized lipids, osteoprotegerin, and bisphosphonates, appear to regulate mineralization in both bone and vasculature and may account for the co-existence of osteoporosis and atherosclerotic calcification that is independent of age.

The epidemiological correlation between osteoporosis and cardiovascular disease is independent of age, but the basis for this correlation is unknown. We previously found that atherogenic oxidized lipids inhibit osteoblastic differentiation in vitro and ex vivo, suggesting that an atherogenic diet may contribute to both diseases. In this study, effects of an atherogenic high‐fat diet versus control chow diet on bone were tested in two strains of mice with genetically different susceptibility to atherosclerosis and lipid oxidation. After 4 months and 7 months on the diets, mineral content and density were measured in excised femurs and lumbar vertebrae using peripheral quantitative computed tomographic (pQCT) scanning. In addition, expression of osteocalcin in marrow isolated from the mice after 4 months on the diets was examined. After 7 months, femoral mineral content in C57BL/6 atherosclerosis‐susceptible mice on the high‐fat diet was 43% lower (0.73 ± 0.09 mg vs. 1.28 ± 0.42 mg; p= 0.008), and mineral density was 15% lower compared with mice on the chow diet. Smaller deficits were observed after 4 months. Vertebral mineral content also was lower in the fat‐fed C57BL/6 mice. These changes in the atherosclerosis‐resistant, C3H/HeJ mice were smaller and mostly not significant. Osteocalcin expression was reduced in the marrow of high fat‐fed C57BL/6 mice. These findings suggest that an atherogenic diet inhibits bone formation by blocking differentiation of osteoblast progenitor cells.

The epidemiological correlation between osteoporosis and cardiovascular disease is independent of age, but the basis for this correlation is unknown. We previously found that atherogenic oxidized lipids inhibit osteoblastic differentiation in vitro and ex vivo, suggesting that an atherogenic diet may contribute to both diseases. In this study, effects of an atherogenic high‐fat diet versus control chow diet on bone were tested in two strains of mice with genetically different susceptibility to atherosclerosis and lipid oxidation. After 4 months and 7 months on the diets, mineral content and density were measured in excised femurs and lumbar vertebrae using peripheral quantitative computed tomographic (pQCT) scanning. In addition, expression of osteocalcin in marrow isolated from the mice after 4 months on the diets was examined. After 7 months, femoral mineral content in C57BL/6 atherosclerosis‐susceptible mice on the high‐fat diet was 43% lower (0.73 ± 0.09 mg vs. 1.28 ± 0.42 mg; p= 0.008), and mineral density was 15% lower compared with mice on the chow diet. Smaller deficits were observed after 4 months. Vertebral mineral content also was lower in the fat‐fed C57BL/6 mice. These changes in the atherosclerosis‐resistant, C3H/HeJ mice were smaller and mostly not significant. Osteocalcin expression was reduced in the marrow of high fat‐fed C57BL/6 mice. These findings suggest that an atherogenic diet inhibits bone formation by blocking differentiation of osteoblast progenitor cells.

The reduced bone mineral density (BMD) observed in osteoporosis results, in part, from reduced activity of bone‐forming osteoblasts. We examined the effect of peroxisome proliferator‐activated receptor (PPAR) activators on MC3T3‐E1 preosteoblast maturation. Activators of PPARα, δ and γ induced alkaline phosphatase activity, matrix calcification and the expression of osteoblast genes as determined by reverse transcriptase‐polymerase chain reaction. However, at relatively high concentrations of the specific PPARγ ligands, ciglitazone and troglitazone, maturation was inhibited. PPARα, δ and γ1 were expressed in MC3T3‐E1 cells. PPARγ1 mRNA and protein levels were induced early during osteoblastic maturation. We speculate that endogenous and pharmacological PPAR activators may affect BMD by modulating osteoblastic maturation.

The reduced bone mineral density (BMD) observed in osteoporosis results, in part, from reduced activity of bone-forming osteoblasts. We examined the effect of peroxisome proliferator-activated receptor (PPAR) activators on MC3T3-E1 preosteoblast maturation. Activators of PPARα, δ and γ induced alkaline phosphatase activity, matrix calcification and the expression of osteoblast genes as determined by reverse transcriptase-polymerase chain reaction. However, at relatively high concentrations of the specific PPARγ ligands, ciglitazone and troglitazone, maturation was inhibited. PPARα, δ and γ1 were expressed in MC3T3-E1 cells. PPARγ1 mRNA and protein levels were induced early during osteoblastic maturation. We speculate that endogenous and pharmacological PPAR activators may affect BMD by modulating osteoblastic maturation.

The cAMP pathway, a major intracellular pathway mediating parathyroid hormone signal, regulates osteoblastic function. Parathyroid hormone (through activation of protein kinase A) has also been shown to stimulate ubiquitin/proteasome activity in osteoblasts. Since the osteoblast-specific transcription factor Osf2/Cbfa1 is important for differentiation of osteoblastic cells, we examined the roles of the cAMP and ubiquitin/proteasome pathways in regulation of Cbfa1. In the osteoblastic cell line, MC3T3-E1, continuous treatment with cAMP elevating agents inhibited both osteoblastic differentiation based on alkaline phosphatase assay and DNA binding ability of Cbfa1 based on a gel retardation assay. Cbfa1 inhibition was paralleled by an inhibitory effect of forskolin on Cbfa1-regulated genes. Northern and Western blot analyses suggested that the inhibition of Cbfa1 by forskolin was mainly at the protein level. Pretreatment with proteasome inhibitors prior to forskolin treatment reversed the effect of forskolin. Furthermore, addition of proteasome inhibitors to forskolin-pretreated samples resulted in recovery of Cbfa1 protein levels and accumulation of polyubiquitinated forms of Cbfa1, indicating a role for the proteasome pathway in the degradation of Cbfa1. These results suggest that suppression of osteoblastic function by the cAMP pathway is through proteolytic degradation of Cbfa1 involving a ubiquitin/proteasome-dependent mechanism.

In osteoporosis, the bone marrow stroma osteogenic cell population declines and adipocyte numbers increase. We recently showed that oxidized lipids inhibit differentiation of preosteoblasts. In this report, we assess the effect of minimally oxidized low density lipoprotein (MM‐LDL) on osteoblastic differentiation of murine marrow stromal cells, M2–10B4. MM‐LDL, but not native LDL, inhibited stromal cell osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, collagen I processing, and mineralization, through a mitogen‐activated protein kinase–dependent pathway. In addition, marrow stromal cells from C57BL/6 mice fed a high fat, atherogenic diet failed to undergo osteogenic differentiation in vitro. The ability of MM‐LDL to regulate adipogenesis was also assessed. Treatment of M2–10B4 as well as 3T3‐L1 preadipocytes with MM‐LDL, but not native LDL, promoted adipogenic differentiation in the presence of peroxisome proliferator‐activated receptor (PPAR) γ agonist thiazolidinediones, BRL49653 and ciglitizone. Based on promoter‐reporter construct experiments, MM‐LDL may be acting in part through activating PPARα. These observations suggest that LDL oxidation products promote osteoporotic loss of bone by directing progenitor marrow stromal cells to undergo adipogenic instead of osteogenic differentiation. These data lend support to the “lipid hypothesis of osteoporosis.”

In osteoporosis, the bone marrow stroma osteogenic cell population declines and adipocyte numbers increase. We recently showed that oxidized lipids inhibit differentiation of preosteoblasts. In this report, we assess the effect of minimally oxidized low density lipoprotein (MM‐LDL) on osteoblastic differentiation of murine marrow stromal cells, M2–10B4. MM‐LDL, but not native LDL, inhibited stromal cell osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, collagen I processing, and mineralization, through a mitogen‐activated protein kinase–dependent pathway. In addition, marrow stromal cells from C57BL/6 mice fed a high fat, atherogenic diet failed to undergo osteogenic differentiation in vitro. The ability of MM‐LDL to regulate adipogenesis was also assessed. Treatment of M2–10B4 as well as 3T3‐L1 preadipocytes with MM‐LDL, but not native LDL, promoted adipogenic differentiation in the presence of peroxisome proliferator‐activated receptor (PPAR) γ agonist thiazolidinediones, BRL49653 and ciglitizone. Based on promoter‐reporter construct experiments, MM‐LDL may be acting in part through activating PPARα. These observations suggest that LDL oxidation products promote osteoporotic loss of bone by directing progenitor marrow stromal cells to undergo adipogenic instead of osteogenic differentiation. These data lend support to the “lipid hypothesis of osteoporosis.”

Although previously believed to be a passive, degenerative process, there is increasing evidence that arterial calcification is actually an active, regulated process. In this review we address potential mechanisms of arterial calcification and, in particular, ways in which it is similar to bone formation. We also advance the hypothesis that particular factors present in atherosclerosis, such as lipids, may explain the co-localization of calcification and atherosclerosis in vivo.

Although revascularization of hypoperfused but metabolically active human myocardium improves segmental function, the temporal relations among restoration of blood flow, normalization of tissue metabolism and recovery of segmental function have not been determined. To examine the effects of coronary angioplasty on 13 asynergic vascular territories in 12 patients, positron emission tomography and two-dimensional echocardiography were performed before and within 72 h of revascularization. Ten patients underwent late echocardiography (67 ± 19 days) and eight underwent a late positron emission tomographic study (68 ±19 days). The extent and severity of abnormalities of wall motion, perfusion and glucose metabolism were expressed as wall motion scores, perfusion defect scores and perfusion-metabolism mismatch scores.

To assess the effect of high intensity ultrasound energy on atherosclerotic arteries, the pressure-volume relation of stenoses in 14 atherosclerotic human cadaver arteries was measured before and after intraarterial application of ultrasound energy. The pressure-volume relation was measured by inflating a 3-mm angioplasty balloon within each artery with use of a syringe equipped with pressure and volume transducers. To minimize potential effects of balloon inflations on compliance, balloon inflation pressure was set at ≤3 atm before ultrasound application. Ultrasound energy was applied by using a titanium wire probe at a frequency of 20 kHz for 2 min.

Although vascular calcification is present in the vast majority of those aged over 70 years and causes serious cardiovascular dysfunction, strategies for its prevention and effective management are currently not available because the mechanism of its pathogenesis is unknown. Recently, similarities between mineralization in the vessel wall and in bone have been recognized. In this review, current understanding of the mechanisms involved in atherosclerotic calcification is summarized. In addition, several observations that may explain the paradox of vascular calcification in the face of osteoporosis are noted, emphasizing the possible role of lipid oxidation products.

Introduction:Although habitual physical activity is associated with improved all-cause mortality in patients with high coronary artery calcification (CAC) scores, higher exercise volume also paradoxically increases CAC in elite endurance athletes.Hypothesis:Based on clinical evidence that parathyroid hormone (PTH) levels are transiently elevated with acute exercise and our previous work demonstrating the effects of intermittent PTH treatment on the microarchitecture of calcific atherosclerotic plaque, we tested whether long-term exercise affects serum PTH levels and the microarchitecture of vascular calcification in mice.Methods:Hyperlipidemic mice (Apoe-/-) were placed on the Western diet to induce basal atherosclerotic calcification. After 20 wks on the diet, the mice were separated (n = 9/group) into a control group and an exercise group that was subjected to an intensive daily treadmill exercise regimen (5x/week) for 9 weeks. Serum collection and microPET/CT imaging were performed at the start and end of the 9-wk period.Results:Serum PTH levels significantly increased 2-fold in the exercise treatment group but remained the same in the control group. MicroCT analysis showed that aortic calcification progressed similarly in both groups, with 25% increase over the 9-wk period. MicroPET analysis revealed that the fold change in 18F-NaF uptake over the 9-wk period was significantly less in the exercise group than the controls, suggesting that the progression of surface area of vascular calcium deposits was attenuated in the exercise group, consistent with more coalescence. Quantitative histological analysis of aortic calcium deposits confirmed that mineral surface area index (MSAi = total calcified perimeter / total calcified area) in the exercise group was less than in the control group (0.09 vs. 0.05; p= 0.076), although this difference did not meet our pre-specified value for statistical significance.Conclusions:Overall, these findings suggest that exercise may shift the microarchitecture of atherosclerotic calcium deposits favorably by promoting coalescence of deposits. Reducing the surface area is expected to reduce compliance mismatch at the interface with soft vascular tissue, which may confer plaque stability.