Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission.

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

Local administration of ibandronate and bone morphogenetic protein-2 after ischemic osteonecrosis of the immature femoral head: a combined therapy that stimulates bone formation and decreases femoral head deformity.

BACKGROUND: Bisphosphonate therapy has been shown to preserve the osteonecrotic femoral head in experimental and short-term clinical studies. However, a lack of new bone formation within the preserved femoral head due to the inhibition of bone remodeling is a concern. The purpose of this investigation was to determine if combined therapy consisting of ibandronate and bone morphogenetic protein-2 (BMP-2) can preserve the shape of the femoral head and stimulate new bone formation in an immature animal model of ischemic osteonecrosis. METHODS: Ischemic osteonecrosis was surgically induced in immature pigs. Four groups were studied: normal, treated with saline solution, treated with ibandronate, and treated with both ibandronate and BMP-2 (the ibandronate + BMP-2 group). The animals were killed eight weeks after surgery. Radiographic, histological, and histomorphometric assessments were performed. RESULTS: Radiographic assessment showed better preservation of the femoral head shape-i.e., a 54% (CI [95% confidence interval]: 22%, 86%) higher mean epiphyseal quotient-in the ibandronate + BMP-2 group than in the saline group. Histological assessment showed increased trabecular bone in the ibandronate + BMP-2 group as compared with that in the saline group. The mean values for trabecular bone volume, thickness, and number and for osteoblast surface were an average of 400% (CI: 242%, 558%), 212% (CI: 166%, 259%), 71% (CI: 6%, 137%), and 2402% (CI: 2113%, 2693%) higher, respectively, in the ibandronate + BMP-2 group than in the saline group. The osteoclast number was significantly reduced in the ibandronate + BMP-2 group compared with that in the saline group (-59% [CI: -75%, -42%]). The mean osteoblast surface value in the ibandronate + BMP-2 group was significantly higher (2567% [CI: 2258%, 2877%]) than that in the ibandronate group. Heterotopic ossifications were present in the capsule of the hip joint in the ibandronate + BMP-2 group. CONCLUSIONS: A combination of ibandronate and BMP-2 decreased femoral head deformity while stimulating bone formation in an immature animal model of ischemic osteonecrosis.

Bisphosphonate-modified gold nanoparticles: a useful vehicle to study the treatment of osteonecrosis of the femoral head.

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of osteonecrosis of the femoral head that presents in children aged 2-14 years. To date, there is no effective medical therapy for treating LCPD largely due to an inability to modulate the repair process, including the predominance of bone resorption. This investigation aims to evaluate the feasibility of using gold nanoparticles (GNPs) that are surface modified with a bisphosphonate compound for the treatment of osteonecrosis at the cellular level. Studies have found osteoclast-mediated resorption to be a process that contributes significantly to the pathogenesis of femoral head deformities arising from Perthes disease. Our in vitro model was designed to elucidate the effect of alendronate-(a bisphosphonate) modified GNPs, on osteoclastogenesis and osteoclast function. RAW 264.7 macrophage cells were cultured with recombinant mouse receptor activator of NF-κB ligand (RANKL), which stimulates osteoclastogenesis, and were then treated with alendronate-modified GNPs for 24, 48, and 72 h. Cell proliferation, osteoclast function, and osteoclast morphology were evaluated by trypan blue dye exclusion assay, tartrate-resistant acid phosphatase (TRAP) staining, and transmission electron microscopy (TEM) imaging. Comparative studies were performed with GNPs that were only stabilized with citrate ions and with alendronate alone. Neither osteoclastogenesis nor osteoclast function were adversely affected by the presence of the citrate-GNP. Alendronate-modified GNPs had an enhanced effect on inducing osteoclast apoptosis and impairing osteoclast function when compared to unbound alendronate populations.

Effects of disruption of epiphyseal vasculature on the proximal femoral growth plate.

BACKGROUND: Proximal femoral growth disturbance is a major complication associated with ischemic osteonecrotic conditions, such as Legg-Calvè-Perthes disease. The extent of ischemic damage and the mechanisms by which ischemic injury to the growing femoral head produces growth disturbance of the proximal femoral growth plate remain unclear. The purpose of this study was to investigate the effects of disruption of the epiphyseal vasculature on the morphology and function of the proximal femoral growth plate in a porcine model. METHODS: Ischemic osteonecrosis of the femoral head was surgically induced in sixty-five piglets by placing a ligature tightly around the femoral neck. Radiographic, histological, micro-computed tomographic, cellular viability, hypoxia marker, and cellular proliferation studies were performed. RESULTS: Disruption of the epiphyseal vasculature did not lead to diffuse growth plate damage in the majority of the ischemic femoral heads. One of the twelve femoral heads analyzed at four weeks and six of the twenty-six femoral heads analyzed at eight weeks had severe disruption of the growth plate that precluded histological assessment of the growth plate zones. In the remaining animals, the proximal part of the femur continued to elongate following induction of ischemia, albeit at a slower rate than on the normal side. Histologically, normal developmental thinning of the growth plate was seen to be absent on the ischemic side. Severe hypoxia and cellular death were limited to the area of the growth plate bordering on the infarcted osseous epiphysis. Normal chondrocytic organization and continued proliferation were observed in the proliferative zone of the growth plate. CONCLUSIONS: In our porcine model, the proximal femoral growth plate was not diffusely damaged following disruption of the epiphyseal vasculature in the majority of the ischemic femoral heads. The majority of the growth plates remained viable and were able to function despite total disruption of the epiphyseal vasculature. These findings suggest that the source of nutrition for the proximal femoral growth plate is not solely the epiphyseal vasculature as has been traditionally believed.

Hypoxia and HIF-1alpha expression in the epiphyseal cartilage following ischemic injury to the immature femoral head.

UNLABELLED: HIF-1alpha has been shown to be a central mediator of cellular response to hypoxia. The role it plays after ischemic injury to the immature femoral head is unknown. The purpose of this study was to determine the region of the femoral head affected by hypoxia following ischemic injury to the immature femoral head and to determine the site of HIF-1alpha activation and revascularization. We hypothesize that the epiphyseal cartilage, rather than the bony epiphysis, is the site of HIF-1alpha activation following ischemic osteonecrosis and that the epiphyseal cartilage plays an important role in the revascularization process. MATERIALS AND METHODS: Femoral head osteonecrosis was surgically induced in 56 immature pigs. Hypoxyprobe staining, cell viability assay, HIF-1alpha western blot, RT-qPCR of HIF-1alpha, VEGF, VEGFR2, and PECAM, and micro-CT assessments of microfil-infused femoral heads were performed. RESULTS: Severe hypoxia was present in the bony epiphysis and the lower part of the epiphyseal cartilage following ischemia. In the bony epiphysis, extensive cell death and tissue necrosis was observed with degradation of proteins and RNAs which precluded further analysis. In the epiphyseal cartilage, the loss of cell viability was limited to its deep layer with the remainder of the cartilage remaining viable. Furthermore, the cartilage from the ischemic side showed a significant increase in HIF-1alpha protein level and HIF-1alpha expression. VEGF expression in the cartilage was dramatically and significantly increased at 24 h, 2 and 4 weeks (p<0.05 for all) with 5 to 10 fold increase being observed on the ischemic side compared to the normal side. PECAM and VEGFR2 expressions in the cartilage were both significantly decreased at 24 h but returned to the normal levels by 2 and 4 weeks, respectively. Micro-CT showed revascularization of the cartilage on the ischemic side with the vessel volume/total volume equaling the normal side by 4 weeks. CONCLUSIONS: Acute ischemic injury to the immature femoral head induced severe hypoxia and cell death in the bony epiphysis and the deep layer of the epiphyseal cartilage. Viable chondrocytes in the superficial layer of the epiphyseal cartilage showed HIF-1alpha activation and VEGF upregulation with subsequent revascularization occurring in the cartilage.

Ischaemic injury to femoral head induces apoptotic and oncotic cell death.

AIMS: The mechanism of cell death in ischaemic osteonecrosis of the femoral head is not clear. Therefore, this study was designed to clarify the mode of cell death following ischaemic osteonecrosis of the femoral head in an established pig model. METHODS: Morphological assessment, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay, detection of DNA laddering and transmission electron microscopy studies were performed to determine whether apoptosis is one of the pathways of cell death following ischaemic osteonecrosis of the femoral head. RESULTS: Mode of cell death was investigated from 2 to 14 days following the surgical induction of ischaemia. Ischaemic femoral heads showed morphological evidence of cell death by oncotic and apoptotic pathways in earlier stages of osteonecrosis. TUNEL positive cells were seen from 2 to 14 days following the induction of ischaemia. DNA samples obtained from ischaemic femoral heads following the induction of ischaemia showed nucleosomal ladders indicating apoptotic cell death. Electron micrographs also showed morphological changes associated with apoptosis. CONCLUSIONS: This study demonstrates that oncosis is not the sole mechanism of cell death following ischaemic injury of the femoral head. Both apoptosis and oncosis are involved as a result of ischaemic injury to the femoral head.

Immunocytochemical localization of atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide in human pancreatic adenocarcinomas.

We recently found that four peptide hormones synthesized by the same gene completely inhibit the growth of human pancreatic adenocarcinomas in athymic mice. The present immunocytochemical investigation was designed to determine where in the adenocarcinomas these peptide hormones localize. Atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide localized to the cytoplasm and nucleus of the human pancreatic adenocarcinomas, which is consistent with their ability to decrease DNA synthesis in the nucleus of this cancer. In this first investigation of where these peptide hormones with anticancer effects localize in any cancer, these peptide hormones also localized to the endothelium of capillaries and fibroblasts within these cancers. This is the first demonstration of growth-inhibiting peptide hormones localizing to the nucleus, where they inhibit DNA synthesis and may interact with growth-promoting hormones that localize there as the etiology of their ability to inhibit the growth of adenocarcinomas both in vitro and in vivo.

Ibandronate for prevention of femoral head deformity after ischemic necrosis of the capital femoral epiphysis in immature pigs.

BACKGROUND: Femoral head deformity is the most serious sequela of ischemic necrosis of the immature femoral head. The purpose of this study was to determine if a highly potent antiresorptive agent, ibandronate, can inhibit bone resorption during the repair of the infarcted femoral head and thus alter the repair process. We hypothesized that preservation of the trabecular framework by inhibiting osteoclastic bone resorption would minimize the development of deformity in a piglet model of ischemic necrosis. The effect of ibandronate on long-bone growth was also assessed. METHODS: Ischemic necrosis of the right femoral head was produced in twenty-four piglets by placing a ligature tightly around the femoral neck. The animals were divided into three groups according to whether they received saline solution, prophylactic treatment, or post-ischemia treatment. The contralateral, untreated femoral heads from the animals that had received saline solution served as the normal control group. At eight weeks, the femoral heads were assessed for deformity with radiography and for trabecular bone indices with histomorphometry. Also, the length of femur from the untreated side was measured on the radiographs and compared among the groups. RESULTS: Radiographic assessment showed that the epiphyseal quotient, determined by dividing the maximum height of the osseous epiphysis by the maximum diameter, was better preserved in the prophylactic (p < 0.001) and post-ischemia (p = 0.02) treatment groups than in the group treated with saline solution. Histomorphometric assessment also showed that the trabecular bone indices were better preserved in the prophylactic and the post-ischemia treatment groups than in the group treated with saline solution (p < 0.01). The mean femoral length on the untreated side of the animals treated with ibandronate was reduced compared with the length on the untreated side of the animals that had received saline solution (p

Increased VEGF expression in the epiphyseal cartilage after ischemic necrosis of the capital femoral epiphysis.

UNLABELLED: Ischemic injury to the immature femoral head produces epiphyseal cartilage damage and cessation of endochondral ossification. This study suggests that VEGF facilitates the repair of the necrotic epiphyseal cartilage, which is essential for restoration of endochondral ossification and re-establishment of the growth of the immature femoral head after ischemic necrosis. INTRODUCTION: Legg-Calve-Perthes disease (LCPD) is a childhood form of osteonecrosis that produces growth arrest of the secondary center of ossification. The cessation of growth is caused by ischemic damage to the hypertrophic zone of the epiphyseal cartilage where endochondral ossification normally occurs. The role of vascular endothelial growth factor (VEGF) in restoring endochondral ossification in the epiphyseal cartilage after ischemic necrosis was investigated in a piglet model of LCPD because the resumption of normal growth is important for maintaining the spherical shape of the femoral head. MATERIALS AND METHODS: Piglet femoral heads were assessed 24 h to 8 weeks after the surgical induction of ischemia. Western blot analysis, ribonuclease protection assay (RPA), immunohistochemistry, and in situ hybridization were performed. RESULTS: Western blot analysis and RPA showed increased VEGF protein and mRNA expression, respectively, in the epiphyseal cartilage of the infarcted heads compared with the contralateral normal heads. In the normal femoral heads, VEGF-immunoreactivity (VEGF-IR) and transcripts were observed in the hypertrophic zone of the epiphyseal cartilage. In the infarcted heads, VEGF-IR and transcripts were no longer observed in the hypertrophic zone because of diffuse cell death in that zone from ischemia. However, VEGF-IR and transcripts were observed in the proliferative zone above the necrotic hypertrophic zone. At 8 weeks, vascular granulation tissue invasion of the necrotic hypertrophic zone was observed with active resorption of the necrotic cartilage. In some areas where the necrotic cartilage was completely resorbed, restoration of endochondral ossification was observed. In these areas, VEGF transcripts were observed in the newly formed hypertrophic zone. CONCLUSIONS: VEGF expression was increased, and its spatial expression was altered in the epiphyseal cartilage after ischemic necrosis of the immature femoral head. VEGF upregulation in the proliferative zone after ischemic damage may play a role in stimulating vascular invasion and granulation tissue formation in the necrotic hypertrophic zone of the epiphyseal cartilage. This may be an important step toward facilitating the resorption of the necrotic cartilage and restoration of endochondral ossification leading to further growth and development of the femoral head.

In vivo cerebrovascular actions of amyloid beta-peptides and the protective effect of conjugated estrogens.

Vascular dysfunction and inflammatory processes may be early events in the pathology of Alzheimer's disease (AD). Even though amyloid beta-peptides (Abeta) play a prominent role in the initiation and progression of cellular dysfunction in AD, the precise in vivo actions of various Abeta-peptides has not been established. The cerebrovascular actions of the major Abeta-peptides (1-40) and (1-42) in live animals were investigated using an open cranial window technique. We show here that the Abeta-peptides cause vascular lesions, especially in the arterioles. In one set of experiments, leukocytes and platelets were tagged with Rhodamine 6G, soluble Abeta(1-40) infused intravenously for 2 minutes, and the vasculature video recorded for 90 minutes. In a second set of experiments, soluble Abeta(1-40) infusion was followed 30 minutes later by an infusion of soluble Abeta(1-42) and the vasculature recorded for 90 minutes. Fluorescent and transmission electron microscopic examinations demonstrated the following cerebrovascular action of Abeta-peptides: endothelial cell damage, leukocyte adhesion, platelet activation, thrombus formation, impeded blood flow, and smooth muscle cell damage. The vascular disruption observed were similar to those observed in the brains of some AD patients and may represent the initial phase of a vascular inflammatory response associated with cerebral amyloid angiopathy. The combination of Abeta(1-40) and (1-42) produced significantly more vascular disruption than Abeta(1-40) alone. Oral administration of conjugated estrogens in ovariectomized female rats protected them from the deleterious actions of Abeta-peptides. The reported protective effect of estrogen against AD may be mediated in part through prevention of cerebrovascular Abeta toxicity.