Hepcidin Deficiency Protects Against Atherosclerosis.

Objective- Inflammatory stimuli enhance the progression of atherosclerotic disease. Inflammation also increases the expression of hepcidin, a hormonal regulator of iron homeostasis, which decreases intestinal iron absorption, reduces serum iron levels and traps iron within macrophages. The role of macrophage iron in the development of atherosclerosis remains incompletely understood. The objective of this study was to investigate the effects of hepcidin deficiency and decreased macrophage iron on the development of atherosclerosis. Approach and Results- Hepcidin- and LDL (low-density lipoprotein) receptor-deficient ( Hamp-/-/ Ldlr-/-) mice and Hamp+/+/ Ldlr-/- control mice were fed a high-fat diet for 21 weeks. Compared with control mice, Hamp-/-/ Ldlr-/- mice had decreased aortic macrophage activity and atherosclerosis. Because hepcidin deficiency is associated with both increased serum iron and decreased macrophage iron, the possibility that increased serum iron was responsible for decreased atherosclerosis in Hamp-/-/ Ldlr-/- mice was considered. Hamp+/+/ Ldlr-/- mice were treated with iron dextran so as to produce a 2-fold increase in serum iron. Increased serum iron did not decrease atherosclerosis in Hamp+/+/ Ldlr-/- mice. Aortic macrophages from Hamp-/-/ Ldlr-/- mice had less labile free iron and exhibited a reduced proinflammatory (M1) phenotype compared with macrophages from Hamp+/+/ Ldlr-/- mice. THP1 human macrophages treated with an iron chelator were used to model hepcidin deficiency in vitro. Treatment with an iron chelator reduced LPS (lipopolysaccharide)-induced M1 phenotypic expression and decreased uptake of oxidized LDL. Conclusions- In summary, in a hyperlipidemic mouse model, hepcidin deficiency was associated with decreased macrophage iron, a reduced aortic macrophage inflammatory phenotype and protection from atherosclerosis. The results indicate that decreasing hepcidin activity, with the resulting decrease in macrophage iron, may prove to be a novel strategy for the treatment of atherosclerosis.

BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy.

Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis.

HDAC9 is implicated in atherosclerotic aortic calcification and affects vascular smooth muscle cell phenotype.

Aortic calcification is an important independent predictor of future cardiovascular events. We performed a genome-wide association meta-analysis to determine SNPs associated with the extent of abdominal aortic calcification (n = 9,417) or descending thoracic aortic calcification (n = 8,422). Two genetic loci, HDAC9 and RAP1GAP, were associated with abdominal aortic calcification at a genome-wide level (P < 5.0 × 10-8). No SNPs were associated with thoracic aortic calcification at the genome-wide threshold. Increased expression of HDAC9 in human aortic smooth muscle cells promoted calcification and reduced contractility, while inhibition of HDAC9 in human aortic smooth muscle cells inhibited calcification and enhanced cell contractility. In matrix Gla protein-deficient mice, a model of human vascular calcification, mice lacking HDAC9 had a 40% reduction in aortic calcification and improved survival. This translational genomic study identifies the first genetic risk locus associated with calcification of the abdominal aorta and describes a previously unknown role for HDAC9 in the development of vascular calcification.

The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth.

The long-term integrity of an articulating joint is dependent upon the nourishment of its cartilage component and the protection of the cartilage surface from friction-induced wear. Loss-of-function mutations in lubricin (a secreted glycoprotein encoded by the gene PRG4) cause the human autosomal recessive disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP). A major feature of CACP is precocious joint failure. In order to delineate the mechanism by which lubricin protects joints, we studied the expression of Prg4 mRNA during mouse joint development, and we created lubricin-mutant mice. Prg4 began to be expressed in surface chondrocytes and synoviocytes after joint cavitation had occurred and remained strongly expressed by these cells postnatally. Mice lacking lubricin were viable and fertile. In the newborn period, their joints appeared normal. As the mice aged, we observed abnormal protein deposits on the cartilage surface and disappearance of underlying superficial zone chondrocytes. In addition to cartilage surface changes and subsequent cartilage deterioration, intimal cells in the synovium surrounding the joint space became hyperplastic, which further contributed to joint failure. Purified or recombinant lubricin inhibited the growth of these synoviocytes in vitro. Tendon and tendon sheath involvement was present in the ankle joints, where morphologic changes and abnormal calcification of these structures were observed. We conclude that lubricin has multiple functions in articulating joints and tendons that include the protection of surfaces and the control of synovial cell growth.

Progesterone receptor membrane component-1 regulates hepcidin biosynthesis.

Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism.

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation.

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Echocardiographic capture of right ventricular wall rupture during inferior wall acute myocardial infarction.

We present the case of a 57-year-old woman with no previous cardiovascular history in whom fatal right ventricular wall rupture was diagnosed by bedside echocardiography early in the management of an inferior wall acute myocardial infarction.

Association between friction and wear in diarthrodial joints lacking lubricin.

OBJECTIVE: The glycoprotein lubricin (encoded by the gene Prg4) is secreted by surface chondrocytes and synovial cells, and has been shown to reduce friction in vitro. In contrast to man-made bearings, mammalian diarthrodial joints must endogenously produce friction-reducing agents. This study was undertaken to investigate whether friction is associated with wear. METHODS: The lubricating ability of synovial fluid (SF) samples from humans with genetic lubricin deficiency was tested in vitro. The coefficient of friction in the knee joints of normal and lubricin-null mice was measured ex vivo; these joints were also studied by light and electron microscopy. Atomic force microscopy was used to image and measure how lubricin reduces friction in vitro. RESULTS: SF lacking lubricin failed to reduce friction in the boundary mode. Joints of lubricin-null mice showed early wear and higher friction than joints from their wild-type counterparts. Lubricin self-organized and reduced the work of adhesion between apposing asperities. CONCLUSION: These data show that friction is coupled with wear at the cartilage surface in vivo. They imply that acquired lubricin degradation occurring in inflammatory joint diseases predisposes the cartilage to damage. Lastly, they suggest that lubricin, or similar biomolecules, will have applications in man-made devices in which reducing friction is essential.

Consequences of disease-causing mutations on lubricin protein synthesis, secretion, and post-translational processing.

Lubricin, a protein product of the gene PRG4, is a secreted mucin-like proteoglycan that is a major lubricant in articulating joints. Mutations in PRG4 cause the autosomal recessive, human disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome. We developed rabbit polyclonal antibodies against human lubricin to determine the consequence of disease-causing mutations at the protein level and to study the protein's normal post-translational processing. Antiserum generated against an epitope in the amino-terminal portion of lubricin detected protein in wild-type synovial fluid and in conditioned media from wild-type cultured synoviocytes. However, the antiserum did not detect lubricin in synovial fluid or cultured synoviocytes from several patients with frameshift or nonsense mutations in PRG4. Antiserum generated against an epitope in the protein's carboxyl-terminal, hemopexin-like domain identified a post-translational cleavage event in wild-type lubricin, mediated by a subtilisin-like proprotein convertase (SPC). Interestingly, in contrast to wild-type lubricin, one disease-causing mutation that removes the last 8 amino acids of the protein, including a conserved cysteine residue, was not cleaved within the hemopexin-like domain when expressed in COS-7 cells. This suggests that formation of an intrachain disulfide bond is required for SPC-mediated cleavage and that SPC-mediated cleavage is essential to protein function.

Mutations in the transmembrane natriuretic peptide receptor NPR-B impair skeletal growth and cause acromesomelic dysplasia, type Maroteaux.

The homodimeric transmembrane receptor natriuretic peptide receptor B (NPR-B [also known as guanylate cyclase B, GC-B, and GUC2B]; gene name NPR2) produces cytoplasmic cyclic GMP from GTP on binding its extracellular ligand, C-type natriuretic peptide (CNP). CNP has previously been implicated in the regulation of skeletal growth in transgenic and knockout mice. The autosomal recessive skeletal dysplasia known as "acromesomelic dysplasia, type Maroteaux" (AMDM) maps to an interval that contains NPR2. We sequenced DNA from 21 families affected by AMDM and found 4 nonsense mutations, 4 frameshift mutations, 2 splice-site mutations, and 11 missense mutations. Molecular modeling was used to examine the putative protein change brought about by each missense mutation. Three missense mutations were tested in a functional assay and were found to have markedly deficient guanylyl cyclase activity. We also found that obligate carriers of NPR2 mutations have heights that are below the mean for matched controls. We conclude that, although NPR-B is expressed in a number of tissues, its major role is in the regulation of skeletal growth.