Collagen XV is necessary for modeling of the extracellular matrix and its deficiency predisposes to cardiomyopathy.

RATIONALE: The extracellular matrix (ECM) is a major determinant of the structural integrity and functional properties of the myocardium in common pathological conditions, and changes in vasculature contribute to cardiac dysfunction. Collagen (Col) XV is preferentially expressed in the ECM of cardiac muscle and microvessels. OBJECTIVE: We aimed to characterize the ECM, cardiovascular function and responses to elevated cardiovascular load in mice lacking Col XV (Col15a1(-/-)) to define its functional role in the vasculature and in age- and hypertension-associated myocardial remodeling. METHODS AND RESULTS: Cardiac structure and vasculature were analyzed by light and electron microscopy. Cardiac function, intraarterial blood pressure, microhemodynamics, and gene expression profiles were studied using echocardiography, telemetry, intravital microscopy, and PCR, respectively. Experimental hypertension was induced with angiotensin II or with a nitric oxide synthesis inhibitor. Under basal conditions, lack of Col XV resulted in increased permeability and impaired microvascular hemodynamics, distinct early-onset and age-dependent defects in heart structure and function, a poorly organized fibrillar collagen matrix with marked interstitial deposition of nonfibrillar protein aggregates, increased tissue stiffness, and irregularly organized cardiomyocytes. In response to experimental hypertension, Col15a1 gene expression was increased in the left ventricle of wild-type mice, and mRNA expression of natriuretic peptides (ANP and BNP) and ECM modeling were abnormal in Col15a1(-/-) mice. CONCLUSIONS: Col XV is necessary for ECM organization in the heart, and for the structure and functions of microvessels. Col XV deficiency leads to a complex cardiac phenotype and predisposes the subject to pathological responses under cardiac stress.

Left ventricular periostin gene expression is associated with fibrogenesis in experimental renal insufficiency.

BACKGROUND: Cardiovascular diseases are the most important cause of death in patients with impaired kidney function. Left ventricular hypertrophy (LVH), cardiac interstitial fibrosis and cardiovascular calcifications are characteristic of chronic renal insufficiency (CRI). Periostin is a fibrogenesis- and calcification-related matricellular protein re-expressed in adult tissues undergoing remodelling in response to pathological stimuli. The role of periostin in CRI-induced LVH is unknown. METHODS: Rats were 5/6-nephrectomized (NX), and after 15 weeks of disease progression high-calcium, high-phosphate or paricalcitol treatment was given for 12 weeks. Cardiac tissue and blood samples were taken to study periostin gene expression and to determine factors contributing to its reactivation, respectively. Left ventricular (LV) periostin expression was also examined in response to angiotensin II or arginine(8)-vasopressin (AVP)-induced pressure overload and in spontaneously hypertensive rats. RESULTS: CRI resulted in a 6.5-fold increase in LV periostin messenger RNA (mRNA) levels. Positive extracellular immunostaining for periostin was detected in areas of infiltrated inflammatory cells and fibrotic lesions. There was a significant correlation between LV periostin mRNA levels and plasma biomarkers of impaired kidney function, LVH, fibrogenesis-related proteins osteopontin and osteoactivin, and anti-calcific matrix Gla protein. Moreover, LV periostin gene expression in CRI correlated positively with systolic blood pressure (BP) and was activated rapidly in response to angiotensin II or AVP infusions. CONCLUSIONS: Periostin is involved in fibrotic cardiac remodelling in CRI. The re-expression of periostin is localized to the fibrotic and inflammatory lesions and is most likely the consequence of elevated BP.

Muscle-derived collagen XIII regulates maturation of the skeletal neuromuscular junction.

Formation, maturation, stabilization, and functional efficacy of the neuromuscular junction (NMJ) are orchestrated by transsynaptic and autocrine signals embedded within the synaptic cleft. Here, we demonstrate that collagen XIII, a nonfibrillar transmembrane collagen, is another such signal. We show that collagen XIII is expressed by muscle and its ectodomain can be proteolytically shed into the extracellular matrix. The collagen XIII protein was found present in the postsynaptic membrane and synaptic basement membrane. To identify a role for collagen XIII at the NMJ, mice were generated lacking this collagen. Morphological and ultrastructural analysis of the NMJ revealed incomplete adhesion of presynaptic and postsynaptic specializations in collagen XIII-deficient mice of both genders. Strikingly, Schwann cells erroneously enwrapped nerve terminals and invaginated into the synaptic cleft, resulting in a decreased contact surface for neurotransmission. Consistent with morphological findings, electrophysiological studies indicated both postsynaptic and presynaptic defects in Col13a1(-/-) mice, such as decreased amplitude of postsynaptic potentials, diminished probabilities of spontaneous release and reduced readily releasable neurotransmitter pool. To identify the role of collagen XIII at the NMJ, shed ectodomain of collagen XIII was applied to cultured myotubes, and it was found to advance acetylcholine receptor (AChR) cluster maturation. Together with the delay in AChR cluster development observed in collagen XIII-deficient mutants in vivo, these results suggest that collagen XIII plays an autocrine role in postsynaptic maturation of the NMJ. Altogether, the results presented here reveal that collagen XIII is a novel muscle-derived cue necessary for the maturation and function of the vertebrate NMJ.

Key roles of endothelin-1 and p38 MAPK in the regulation of atrial stretch response.

Mechanisms regulating stretch response in the left ventricle are investigated in detail but not well understood in atrial myocardium. Hypertrophic growth of atrial myocardium contributes to the pathogenesis of atrial fibrillation. In this study, we sought to elucidate mechanisms of stretch-induced activation of key signaling pathways and hypertrophy-associated genes in rat atria. Stretching of isolated atria induced a rapid increase in phosphorylation of p38 MAPK and ERK and induced a p38 MAPK-dependent increase in DNA binding activity of transcription factors Elk-1 and GATA-4. Inhibition of the ERK pathway had no effect on the cardiac transcription factors studied. Stretch-induced increase in atrial contractile function was substantially enhanced by inhibition of p38 MAPK. p38 MAPK also regulated stretch-induced increase in c-fos, ß-myosin heavy chain, B-type natriuretic peptide mRNA levels, and atrial natriuretic peptide secretion in isolated atria. Various autocrine/paracrine factors are known to mediate the stretch response in the left ventricle. Stretching of isolated atria resulted in a robust increase in endothelin-1 (ET-1) mRNA levels, while apelin and adrenomedullin signaling cascades were downregulated. Administration of mixed ET(A/B) receptor antagonist bosentan attenuated the stretch-induced activation of GATA-4 in isolated atria, whereas ANG II receptor type-1 antagonist CV-11974 had no effect. Moreover, analysis of RNA from intact atrial and ventricular myocardium revealed significantly higher mRNA levels of ET(A) receptor and ET converting enzyme-1 in atrial compared with ventricular myocardium. In conclusion, our findings identify the local ET-1 system and p38 MAPK as key regulators of load-induced hypertrophic response in isolated rat atria.

BMP-4 expression has prognostic significance in advanced serous ovarian carcinoma and is affected by cisplatin in OVCAR-3 cells.

Several angiogenesis-promoting factors have prognostic significance in ovarian cancer. The objective of this study was to evaluate whether traditional chemotherapy affects angiogenesis-related factors in ovarian carcinoma and to assess the clinical significance of these effects. To screen for angiogenesis-related factors of possible relevance, OVCAR-3 and A2780 ovarian cancer cells were treated with IC(50) doses of cisplatin (CDDP) or docetaxel, or with bevacizumab, and mRNA expression of several angiogenesis-related factors was analyzed. Thrombospondin-1 (TSP-1), bone morphogenetic protein-4 (BMP-4), endothelin-1, and placental growth factor-2 were statistically significantly induced by CDDP. At protein level, CDDP also induced hypoxia-inducible factor-1α but not vascular endothelial growth factor. In carcinoma samples taken before and after platinum-based neoadjuvant chemotherapy from 28 patients with advanced, high-grade serous ovarian carcinoma, CD105 and factors most induced by CDDP (TSP-1 and BMP-4) were analyzed by immunohistochemistry. Strong expression of BMP-4 before chemotherapy was an independent prognostic factor of longer progression-free time (p = 0.002) and overall survival (p = 0.02), but it was not associated with neovascularization (as evaluated by CD105). However, changes in BMP-4 expression in samples analyzed before and after chemotherapy (observed in 22/28 patients) were not associated with prognosis. TSP-1 expression was not associated with clinical parameters. Our results indicate that in serous ovarian carcinoma, BMP-4 has prognostic significance, which is not angiogenesis-related. We also show that CDDP induces several angiogenesis-related growth factors in vitro and future studies are warranted to clarify the clinical significance of this phenomenon.

Pemphigoid gestationis autoantigen, transmembrane collagen XVII, promotes the migration of cytotrophoblastic cells of placenta and is a structural component of fetal membranes.

In pemphigoid gestationis (PG), autoantibodies target collagen XVII, a hemidesmosomal transmembrane protein, which is an important element in cutaneous epithelial adhesion and signalling. We report that collagen XVII is expressed in the first trimester and term syncytial and cytotrophoblastic cells of normal placenta and in epithelial cells of amniotic membrane. Immunoelectron microscopy confirmed the localization of collagen XVII to the hemidesmosomes of amniotic epithelium. Examination of three PG placentas showed mild villitis, but there were no differences between collagen XVII expression levels or immunostaining signals as compared to normal placenta. Collagen XVII expression was also detected in cultured extravillous trophoblast HTR-8/SVneo cells, where collagen XVII expression was upregulated by PMA and TGF-beta1. Interestingly, the presence of Col15, the cell migration domain of collagen XVII, induced the migration of HTR-8/SVneo cells in transmigration assay. Analysis of amniotic fluid samples at different gestational weeks revealed that a large quantity of collagen XVII ectodomain was shed into amniotic fluid throughout pregnancy. Biochemical and immunoblotting analysis indicated that the ectodomain in amniotic fluid is structurally very similar to the ectodomain produced by cultured keratinocytes. Cultured cells from amniotic fluid samples also expressed collagen XVII. Our results suggest that collagen XVII may contribute to the invasion of extravillous trophoblasts during placental development and is also required for the integrity of amniotic basement membrane. Although the exact pathomechanism of PG is still largely unknown, the clinical symptoms of PG are initiated after the expression of collagen XVII in placenta during the first trimester of pregnancy.

Thrombospondin-4 expression is rapidly upregulated by cardiac overload.

The precise mechanisms regulating gene expression of thrombospondins (TSPs) in the heart remain incompletely understood. Here we characterized cardiac TSP-4 expression in response to pressure overload and myocardial infarction in vivo. Arginine(8)-vasopressin (AVP) infusion increased left ventricular (LV) TSP-4 mRNA levels within 30 min. Also angiotensin II infusion rapidly activated LV TSP-4 expression, TSP-4 mRNA levels being highest at 6h and protein at 72 h and 2 weeks. During remodeling process following myocardial infarction, LV TSP-4 mRNA levels increased at day one, as studied by quantitative RT-PCR. TSP-4 immunostaining was localized to endothelial cells in hypertrophied hearts of spontaneously hypertensive rats. AVP-infusion increased LV TSP-1 mRNA levels similarly to TSP-4 within 30 min showing that rapid induction of gene expression, well before the development of cardiac hypertrophy, is typical for the thrombospondin family. These results further suggest that TSP-4 may be an endothelial specific marker of cardiac overload.

ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta.

We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of (14)C-PhIP (2 microM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72+/-0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of (14)C-PhIP from maternal to fetal circulation (FM ratio 0.90+/-0.08 at 6 h, p<0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75+/-0.10, p=0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of (14)C-PhIP (R=-0.81, p<0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP (R: -0.11, p=0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of (14)C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarcinoma cells.

Identification of cell cycle regulatory and inflammatory genes as predominant targets of p38 mitogen-activated protein kinase in the heart.

Mitogen-activated protein kinases (MAPKs) regulate cardiomyocyte growth and apoptosis in response to extracellular stimulation, but the downstream effectors that mediate their pathophysiological effects remain poorly understood. We determined the targets and role of p38 MAPK in the heart in vivo by using local adenovirus-mediated gene transfer of constitutively active upstream kinase mitogen-activated protein kinase kinase 3b (MKK3bE) and wild-type p38alpha in rats. DNA microarray analysis of animals with cardiac-specific overexpression of p38 MAPK revealed that 264 genes were upregulated more than 2-fold including multiple genes controlling cell division, cell signaling, inflammation, adhesion, and transcription. A large number of previously unknown p38 target genes were found. Using gel mobility-shift assays we identified several cardiac transcription factors that were directly activated by p38 MAPK. Finally, we determined the functional significance of the altered cardiac gene-expression profile by histological analysis and echocardiographic measurements, which indicated that p38 MAPK overexpression-induced gene expression results in myocardial cell proliferation, inflammation, and fibrosis. In conclusion, we defined the novel target genes and transcription factors as well as the functional effects of p38 MAPK in the heart. Expression profiling of p38 MAPK overexpression identified cell cycle regulatory and inflammatory genes critical for pathological processes in the adult heart.

Nuclear factor-kappaB signaling contributes to severe, but not moderate, angiotensin II-induced left ventricular remodeling.

OBJECTIVE: The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated in cardiomyocyte hypertrophy in vitro as well as in vivo; however, it is unknown if activation of NF-kappaB plays a mandatory role in the hypertrophic process. Here we characterize the importance of NF-kappaB signaling in moderate and severe left ventricular (LV) hypertrophy in rats with chronic pressure overload induced by angiotensin II (Ang II) infusion. METHODS AND RESULTS: Electrophoretic mobility shift assay analysis revealed that Ang II infusion (2.5 microg/kg per min) for 6 days increased LV NF-kappaB/DNA-binding activity in a biphasic manner in Sprague-Dawley rats. Pyrrolidine dithiocarbamate (PDTC) (100 mg/kg per day), an NF-kappaB inhibitor, abolished Ang II-induced NF-kappaB activation and concomitant increase in tumor necrosis factor-alpha gene expression, while activator protein-1/DNA binding was not affected. Inhibition of NF-kappaB signaling for 6 days significantly attenuated Ang II-induced increases in LV/body weight ratio, LV mean wall thickness and cardiomyocyte cross-sectional area, without compromising LV systolic function. Moreover, PDTC abolished Ang II-induced cardiomyocyte apoptosis and interstitial fibrosis, and attenuated the gene expression of type I collagen. In contrast, a moderate LV hypertrophy induced by Ang II at a lower dose (0.5 microg/kg per min) was not associated with a significant activation of NF-kappaB, and PDTC treatment had no effect on the hypertrophic indices. CONCLUSION: Our in-vivo data indicate a critical role of NF-kappaB signaling in the advanced stage of the remodeling process, whereas development of moderate LV hypertrophy is not dependent on NF-kappaB activation.

p38 Kinase rescues failing myocardium after myocardial infarction: evidence for angiogenic and anti-apoptotic mechanisms.

As a leading cause of heart failure, postinfarction left ventricular remodeling represents an important target for therapeutic interventions. Mitogen-activated protein kinases regulate critical cellular processes including stress response and survival, but their role in left ventricular remodeling is unknown. In the present study, rats were subjected to myocardial infarction by ligating the left anterior descending coronary artery. Western blot and kinase assay analysis revealed an inactivation of p38 kinase after myocardial infarction. Local adenovirus-mediated cotransfection of wild-type (WT) p38 kinase and constitutively active MKK3b reduced infarct size (26+/-3% vs. 47+/-4%, P<0.05 vs. LacZ-treated control) associated with improved ejection fraction (66.9+/-5.5% vs. 44.4+/-4.0%, P<0.001), fractional shortening (30.2+/-2.1% vs. 19.7+/-2.2%, P<0.001), and decreased left ventricular diastolic diameter (8.5+/-0.4 mm vs. 9.5+/-0.2 mm, P<0.01). p38 kinase gene transfer increased capillary density (2423+/-107/mm(2) vs. 1934+/-86/mm(2), P<0.001) and resulted in microvessel enlargement in the ischemic border zone. Apoptosis (35+/-7 vs. 69+/-13 cells, P<0.01) and fibrosis (16+/-3% vs. 34+/-8%, P<0.05) were reduced, while the number of c-kit positive cardiac stem-like cells remained unchanged. These results indicate that reduced p38 signaling predisposes to adverse postinfarction remodeling. The rescue of failing myocardium with p38 kinase may be a potential new therapy for heart failure after myocardial infarction.

Expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinase-2 and -9 in horses with chronic airway inflammation.

OBJECTIVE To examine whether expression of extracellular matrix metalloproteinase inducer (EMMPRIN) can be detected in equine lungs and whether it correlates with matrix metalloproteinase (MMP)-2 and -9 expression in bronchoalveolar lavage fluid (BALF) of horses with chronic inflammation of the lungs (ie, lower airway inflammation [LAI]). ANIMALS 29 horses with signs of chronic respiratory tract disease, which were classified as the LAI (n = 17) and LAI with respiratory distress (RDLAI [12]) groups, and 15 control horses. PROCEDURES BALF, tracheal aspirate, and blood samples were obtained, and EMMPRIN expression was determined from BALF cells and RBCs by use of western blotting. Activities of MMP-2 and -9 were determined with zymography. RESULTS Expression of EMMPRIN protein was identified in BALF cells of all horses. Expression of EMMPRIN protein was highest for the RDLAI group and was correlated with MMP-2 and -9 protein expression, MMP-9 gelatinolytic activity, and airway neutrophilia. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that EMMPRIN was involved in the pathophysiologic processes of asthma in horses. However, additional studies of horses and other species are warranted to elucidate the regulation of EMMPRIN expression in asthmatic lungs.

Crosstalk between Jagged1 and GDNF/Ret/GFRalpha1 signalling regulates ureteric budding and branching.

Glial-Cell-Line-Derived Neurotrophic Factor (GDNF) is the major mesenchyme-derived regulator of ureteric budding and branching during nephrogenesis. The ligand activates on the ureteric bud epithelium a receptor complex composed of Ret and GFRalpha1. The upstream regulators of the GDNF receptors are poorly known. A Notch ligand, Jagged1 (Jag1), co-localises with GDNF and its receptors during early kidney morphogenesis. In this study we utilized both in vitro and in vivo models to study the possible regulatory relationship of Ret and Notch pathways. Urogenital blocks were exposed to exogenous GDNF, which promotes supernumerary ureteric budding from the Wolffian duct. GDNF-induced ectopic buds expressed Jag1, which suggests that GDNF can, directly or indirectly, up-regulate Jag1 through Ret/GFRalpha1 signalling. We then studied the role of Jag1 in nephrogenesis by transgenic mice constitutively expressing human Jag1 in Wolffian duct and its derivatives under HoxB7 promoter. Jag1 transgenic mice showed a spectrum of renal defects ranging from aplasia to hypoplasia. Ret and GFRalpha1 are normally downregulated in the Wolffian duct, but they were persistently expressed in the entire transgenic duct. Simultaneously, GDNF expression remained unexpectedly low in the metanephric mesenchyme. In vitro, exogenous GDNF restored the budding and branching defects in transgenic urogenital blocks. Renal differentiation apparently failed because of perturbed stimulation of primary ureteric budding and subsequent branching. Thus, the data provide evidence for a novel crosstalk between Notch and Ret/GFRalpha1 signalling during early nephrogenesis.

Abnormal maturation of the retinal vasculature in type XVIII collagen/endostatin deficient mice and changes in retinal glial cells due to lack of collagen types XV and XVIII.

Type XVIII collagen is important in the early phase of retinal vascular development and for the regression of the primary vasculature in the vitreous body after birth. We show here that the retina in Col18a1-/- mice becomes densely vascularized by anomalous anastomoses from the persistent hyaloid vasculature by day 10 after birth. In situ hybridizations revealed normal VEGF mRNA expression, but the phenotype of collagen XVIII deficient mice closely resembled that of mice expressing VEGF120 and VEGF188 isoforms only, suggesting that type XVIII collagen may be involved in VEGF function. Type XVIII collagen was found to be indispensable for angiogenesis in the eye, as also oxygen-induced neovascularization was less intense than normal in the Col18a1-/- mice. We observed a marked increase in the amount of retinal astrocytes in the Col18a1-/- mice. Whereas the retinal vessels of wild-type mice are covered by astrocytes and the regressing, thin hyaloid vessels are devoid of astrocytes, the retinal vessels in the Col18a1-/- mice were similarly covered by astrocytes but not the persistent hyaloid vessels in the vitreous body. Interestingly, double null mice lacking type XVIII collagen and its homologue type XV collagen had the persistent hyaloid vessels covered by astrocytes, including the parts located in the vitreous body. We thus hypothesize that type XV collagen is a regulator of glial cell recruitment around vessels and that type XVIII collagen regulates their proliferation.

Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility.

The orphan receptor APJ and its recently identified endogenous ligand, apelin, exhibit high levels of mRNA expression in the heart. However, the functional importance of apelin in the cardiovascular system is not known. In isolated perfused rat hearts, infusion of apelin (0.01 to 10 nmol/L) induced a dose-dependent positive inotropic effect (EC50: 33.1+/-1.5 pmol/L). Moreover, preload-induced increase in dP/dt(max) was significantly augmented (P<0.05) in the presence of apelin. Inhibition of phospholipase C (PLC) with U-73122 and suppression of protein kinase C (PKC) with staurosporine and GF-109203X markedly attenuated the apelin-induced inotropic effect (P<0.001). In addition, zoniporide, a selective inhibitor of Na+-H+ exchange (NHE) isoform-1, and KB-R7943, a potent inhibitor of the reverse mode Na+-Ca2+ exchange (NCX), significantly suppressed the response to apelin (P<0.001). Perforated patch-clamp recordings showed that apelin did not modulate L-type Ca2+ current or voltage-activated K+ currents in isolated adult rat ventricular myocytes. Apelin mRNA was markedly downregulated in cultured neonatal rat ventricular myocytes subjected to mechanical stretch and in vivo in two models of chronic ventricular pressure overload. The present study provides the first evidence for the physiological significance of apelin in the heart. Our results show that apelin is one of the most potent endogenous positive inotropic substances yet identified and that the inotropic response to apelin may involve activation of PLC, PKC, and sarcolemmal NHE and NCX.

Type XIII collagen strongly affects bone formation in transgenic mice.

UNLABELLED: To characterize the function of type XIII collagen, a transmembrane protein occurring at cell adhesion sites, we generated transgenic mice overexpressing it. High transgene expression was detected in cartilage and bone. The overexpression mice developed an unexpected skeletal phenotype marked by a massive increase in bone mass caused by increased bone formation rather than impaired resorption. INTRODUCTION: Type XIII collagen is a type II transmembrane protein that is expressed in many tissues throughout development and adult life. It is located in focal adhesions of cultured fibroblasts and other cells and in the adhesive structures of tissues. To further characterize the function of this protein, we generated transgenic mice overexpressing it. High transgene expression was detected in cartilage and bone in locations also containing the endogenous protein. MATERIALS AND METHODS: Col13a1 5'-flanking sequences were tested for their efficiencies to drive gene expression. Skeletal tissues of transgenic mice and wildtype littermates were compared using histological, immunohistochemical, and bone histomorphometrical analyses. Bone formation rate was measured by tetracycline double-labeling. Osteoclast number and resorption activity were determined using standard methods. RNA samples from transgenic and wildtype femurs were analyzed by Northern blotting and quantitative RT-PCR. RESULTS: There was no defect in early skeletal development, but the high bone mass phenotype became apparent in heterozygous mice at the age of 3-4 weeks. The changes were most noticeable in proximal long bones but were also detectable in calvarial bones. The cortical bone cross-sectional area and the volumetric BMD were highly increased, but the bone marrow was well formed. Histological and histomorphometric analysis showed that trabecular bone volume was not significantly altered. Because of the normal epiphyseal growth plates, the longitudinal growth was not affected. Bone formation rate was several times higher in the overexpression mice than in their normal littermates, whereas the osteoclast number and resorption activity were normal. RNA analysis revealed increased expression in the transcription factor Runx2 and IGF-II, both known to be involved in bone biology. CONCLUSION: Overexpression of type XIII collagen in skeletal tissues leads postnatally to an abnormally high bone mass caused by increased bone formation rather than impaired resorption. The findings suggest that type XIII collagen has an important role in bone modeling, and in particular, it may have a function in coupling the regulation of bone mass to mechanical use.

Evidence for a functional role of angiotensin II type 2 receptor in the cardiac hypertrophic process in vivo in the rat heart.

BACKGROUND: The precise function of angiotensin II type 2 receptor (AT2-R) in the mammalian heart in vivo is unknown. Here, we investigated the role of AT2-R in cardiac pressure overload. METHODS AND RESULTS: Rats were infused with vehicle, angiotensin II (Ang II), PD123319 (an AT2-R antagonist), or the combination of Ang II and PD123319 via subcutaneously implanted osmotic minipumps for 12 or 72 hours. Ang II-induced increases in mean arterial pressure, left ventricular weight/body weight ratio, and elevation of skeletal alpha-actin and beta-myosin heavy chain mRNA levels were not altered by PD123319. In contrast, AT2-R blockade resulted in a marked increase in the gene expression of c-fos, endothelin-1, and insulin-like growth factor-1 in Ang II-induced hypertension. In parallel, Ang II-stimulated mRNA and protein expression of atrial natriuretic peptide were significantly augmented by AT2-R blockade. Moreover, PD123319 markedly increased the synthesis of B-type natriuretic peptide. Furthermore, the expression of vascular endothelial growth factor and fibroblast growth factor-1 was downregulated by Ang II only in the presence of AT2-R blockade. CONCLUSIONS: Our results provide evidence that AT2-R plays a functional role in the cardiac hypertrophic process in vivo by selectively regulating the expression of growth-promoting and growth-inhibiting factors.

The expression of MT1 and MT2 melatonin receptor mRNA in several rat tissues.

The mechanisms that mediate the various effects of melatonin in mammalian tissues are not always known. Therefore, the aim of this study was to investigate whether MT(1) and MT(2) melatonin receptors are expressed in certain tissues of the rat. The expression of MT(1) and MT(2) melatonin receptor mRNA was determined using a real-time quantitative RT-PCR method. In addition, we examined whether mRNA for either subtype of receptor shows any difference in the expression between midnight and noon, similar to the changes in melatonin concentrations in plasma and tissue samples. MT(1) and MT(2) melatonin receptor mRNAs were found in the rat hypothalamus, retina and small intestine. We also showed a low expression of MT(2) mRNA in the rat liver and heart SA node. In the heart apex and the Harderian gland, no appearance of either of the receptor mRNAs was detectable. A significant difference in the expression of MT(1) mRNA between day and night was found in the hypothalamus. In conclusion, our findings suggest that at least some effects of melatonin are mediated through membrane MT(1) and MT(2) receptors in the hypothalamus, the retina and the small intestine. Down-regulation of receptors might be one reason for the difference in the hypothalamic MT(1) melatonin receptor mRNA expression between midnight and noon. In the liver and the heart SA node, the physiological significance of possible MT(2) receptors remains unclear. According to our negative midnight and noon results in the Harderian gland and heart apex melatonin may exert its effect on these tissues by a non-receptor mechanism.

Expression of monocarboxylate transporters I and IV and the ancillary protein CD147 in the intestinal tract of healthy horses and ponies.

OBJECTIVE: To characterize the expression of monocarboxylate transporters (MCTs) 1 and 4 and the ancillary protein CD147 in the intestinal tract of healthy equids and determine the cellular location of CD147 in the intestinal epithelium. ANIMALS: 12 healthy horses and ponies slaughtered for meat production or euthanized for reasons unrelated to gastrointestinal tract disease. PROCEDURES: The entire gastrointestinal tract was removed from each equid within 45 minutes after slaughter or euthanasia. Tissue samples were obtained from the antimesenteric side of the duodenum, jejunum, ileum, middle part of the cecum, sternal flexure of the ventral colon, pelvic flexure, sternal flexure of the dorsal colon, and descending colon (small colon). Expressions of MCT1, MCT4, and the ancillary protein CD147 were examined in tissue samples from each of the 8 intestinal locations by means of quantitative PCR assay, immunoblotting, and immunohistochemical analyses. RESULTS: Expression of MCT1 was most abundant in the cecum and colonic sites, whereas expression of MCT4 was predominantly in the proximal section of the intestine (small intestinal sites and cecum). Immunohistochemical analysis revealed that MCT1 and CD147 were present in the membranes of enterocytes (in crypts and villi). CONCLUSIONS AND CLINICAL RELEVANCE: The anatomic distribution of MCT1 and MCT4 in the equine intestinal tract determined in this study together with the previous knowledge of the sites of substrate absorption indicated that MCT1 might predominantly contribute to the uptake of short-chain fatty acids in the large intestine and MCT4 might predominantly contribute to the uptake of lactate in the small intestine.

Bone morphogenetic protein-2--a potential autocrine/paracrine factor in mediating the stretch activated B-type and atrial natriuretic peptide expression in cardiac myocytes.

Hemodynamic overload exposes the heart to variety of neural, humoral and mechanical stresses. Even without the neurohumoral control of the entire organism cardiac myocytes have the ability to sense mechanical stretch and convert it into adaptive intracellular signals. This process is controlled by several growth factors. Here we show that mechanical stretch in vitro and hemodynamic overload in vivo activated the expression of bone morphogenetic protein-2 (BMP-2), while expression of BMP-4 was temporarily attenuated by stretch. BMP-2 and BMP-4 alone stimulated B-type and atrial natriuretic peptide (BNP and ANP) expression and protein synthesis, and activated transcription factor GATA-4 resembling the effects of mechanical stretch of cultured cardiac myocytes. Further, BMP antagonist Noggin was able to inhibit stretch and hypertrophic agonist induced BNP and ANP expression. Together these data provide evidence for BMP-2 as a new autocrine/paracrine factor that regulates cardiomyocyte mechanotransduction and adaptation to increased mechanical stretch.