Toll-like receptor 4 mediates vascular remodeling in hyperhomocysteinemia

Nephrotic syndrome (NS) is a kidney disease predominantly present in children with idiopathic condition; final stage of the disease progresses into end-stage renal disease. Generally, NS is treated using standard steroid therapy, however; most of the children are steroid sensitive and about 15–20% are non-responders (SRNS). Non-responsiveness of these children would be a risk with the possibility of mutational changes in podocyte genes (NPHS1, NPHS2, WT1, PLCE1). The mutation in podocyte genes is associated with SRNS. NPHS1, NPHS2, and WT1 genes are identified/directly linked to SRNS. The present study is a surveillance on the mutation analysis of WT1 (exons 8 and 9) and NPHS2 (exons 1–8) gene in SRNS followed by clinical management. In the present study, we analyzed these two genes in a total of 117 SRNS (73 boys and 44 girls) children. A total of five mutations were detected in six children. First, WT1 mutation was detected at 9th intron-IVS 9 + 4C > T position in one SRNS female patient. This WT1 mutation was identified in a girl having Frasier Syndrome (FS) with focal segmental glomerulosclerosis and a complete sex reversal found through molecular and karyological screening. In NPHS2, missense mutations of P20L (in two children), P316S, and p.R229Q, and a frame shift mutation of 42delG were detected. Thus, applying molecular investigation helped us to decide on treatment plan of SRNS patients, mainly to avoid unnecessary immunosuppressive treatment.     

Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia

Cardiomyocyte N-methyl-d-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (-dL/dt), and maximal rate of relaxation (+dL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, -dL/dt, and +dL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1.     

Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia

An elevated level of homocysteine (Hcy) limits the growth and induces apoptosis. However, the mechanism of Hcy-induced programmed cell death in endothelial cells is largely unknown. We hypothesize that Hcy induces intracellular reactive oxygen species (ROS) production that leads to the loss of transmembrane mitochondrial potential (Deltapsi(m)) accompanied by the release of cytochrome-c from mitochondria. Cytochrome-c release contributes to caspase activation, such as caspase-9, caspase-6, and caspase-3, which results in the degradation of numerous nuclear proteins including poly (ADP-ribose) polymerase (PARP), which subsequently leads to the internucleosomal cleavage of DNA, resulting cell death. In this study, rat heart microvascular endothelial cells (MVEC) were treated with different doses of Hcy at different time intervals. Apoptosis was measured by DNA laddering and transferase-mediated dUTP nick-end labeling (TUNEL) assay. ROS production and MP were determined using fluorescent probes (2,7-dichlorofluorescein (DCFH-DA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide (JC-1), respectively, by confocal microscopy. Differential gene expression for apoptosis was analyzed by cDNA array. The results showed that Hcy-mediated ROS production preceded the loss of MP, the release of cytochrome-c, and the activation of caspase-9 and -3. Moreover the Hcy treatment resulted in a decrease in Bcl(2)/Bax ratio, evaluated by mRNA levels. Caspase-9 and -3 were activated, causing cleavage of PARP, a hallmark of apoptosis and internucleosomal DNA fragmentation. The cytotoxic effect of Hcy was blocked by using small interfering RNA (siRNA)-mediated suppression of caspase-9 in MVEC. Suppressing the activation of caspase-9 inhibited the activation of caspase -3 and enhanced the cell viability and MP. Our data suggested that Hcy-mediated ROS production promotes endothelial cell death in part by disturbing MP, which results in subsequent release of cytochrome-c and activation of caspase-9 and 3, leading to cell death.     

Increased intracortical bone remodeling during lactation in beagle dogs

There are substantial changes in skeletal and mineral metabolism during pregnancy and lactation. The purpose of this study was to determine the changes in intracortical bone remodeling and turnover during lactation in beagle dogs. A femur and rib were obtained from dogs near the end of lactation or soon after weaning and compared with nonlactating controls. Rib cortical bone had much higher bone turnover rates than did femoral diaphyseal cortical bone. The number of single-labeled osteons and the number of resorption spaces were significantly greater during lactation in both the rib and the femur. Additionally, the mineral apposition rate, basic multicellular unit activation frequency, and bone turnover rates were greater in the femoral cortical bone from the lactating dogs than from the controls. These data demonstrate that during lactation, intracortical bone remodeling increases, and this may provide a mechanism for the skeleton to be responsive to the calcium requirements of the mother. In addition, these data may help explain the transient decreases in cortical bone mineral density that are reported to occur during human lactation.     

Mitochondrial regulation of epigenetics and its role in human diseases

Most pathogenic mitochondrial DNA (mtDNA) mutations induce defects in mitochondrial oxidative phosphorylation (OXPHOS). However, phenotypic effects of these mutations show a large degree of variation depending on the tissue affected. These differences are difficult to reconcile with OXPHOS as the sole pathogenic factor suggesting that additional mechanisms contribute to lack of genotype and clinical phenotype correlationship. An increasing number of studies have identified a possible effect on the epigenetic landscape of the nuclear genome as a consequence of mitochondrial dysfunction. In particular, these studies demonstrate reversible or irreversible changes in genomic DNA methylation profiles of the nuclear genome. Here we review how mitochondria damage checkpoint (mitocheckpoint) induces epigenetic changes in the nucleus. Persistent pathogenic mutations in mtDNA may also lead to epigenetic changes causing genomic instability in the nuclear genome. We propose that “mitocheckpoint” mediated epigenetic and genetic changes may play key roles in phenotypic variation related to mitochondrial diseases or host of human diseases in which mitochondrial defect plays a primary role.     

Mitochondrial mechanism of oxidative stress and systemic hypertension in hyperhomocysteinemia

Formation of homocysteine (Hcy) is the constitutive process of gene methylation. Hcy is primarily synthesized by de-methylation of methionine, in which s-adenosyl-methionine (SAM) is converted to s-adenosyl-homocysteine (SAH) by methyltransferase (MT). SAH is then hydrolyzed to Hcy and adenosine by SAH-hydrolase (SAHH). The accumulation of Hcy leads to increased cellular oxidative stress in which mitochondrial thioredoxin, and peroxiredoxin are decreased and NADH oxidase activity is increased. In this process, Ca2+-dependent mitochondrial nitric oxide synthase (mtNOS) and calpain are induced which lead to cytoskeletal de-arrangement and cellular remodeling. This process generates peroxinitrite and nitrotyrosine in contractile proteins which causes vascular dysfunction. Chronic exposure to Hcy instigates endothelial and vascular dysfunction and increases vascular resistance causing systemic hypertension. To compensate, the heart increases its load which creates adverse cardiac remodeling in which the elastin/collagen ratio is reduced, causing cardiac stiffness and diastolic heart failure in hyperhomocysteinemia.     

Mitochondrial regulation of epigenetics and its role in human diseases

Most pathogenic mitochondrial DNA (mtDNA) mutations induce defects in mitochondrial oxidative phosphorylation (OXPHOS). However, phenotypic effects of these mutations show a large degree of variation depending on the tissue affected. These differences are difficult to reconcile with OXPHOS as the sole pathogenic factor suggesting that additional mechanisms contribute to lack of genotype and clinical phenotype correlationship. An increasing number of studies have identified a possible effect on the epigenetic landscape of the nuclear genome as a consequence of mitochondrial dysfunction. In particular, these studies demonstrate reversible or irreversible changes in genomic DNA methylation profiles of the nuclear genome. Here we review how mitochondria damage checkpoint (mitocheckpoint) induces epigenetic changes in the nucleus. Persistent pathogenic mutations in mtDNA may also lead to epigenetic changes causing genomic instability in the nuclear genome. We propose that “mitocheckpoint” mediated epigenetic and genetic changes may play key roles in phenotypic variation related to mitochondrial diseases or host of human diseases in which mitochondrial defect plays a primary role.     

GABA receptors and nitric oxide ameliorate constrictive collagen remodeling in hyperhomocysteinemia

Elevated plasma levels of homocysteine (Hcy) are associated with vascular dementias and Alzheimer's disease. The role of Hcy in brain microvascular endothelial cell (MVEC) remodeling is unclear. Hcy competes with muscimol, an gamma-amino butyric acid (GABA)-A receptor agonist. GABA is the primary inhibitory neurotransmitter in the brain. Our hypothesis is that Hcy induces constrictive microvascular remodeling by altering GABA-A/B receptors. MVEC from wild type, matrix metalloproteinase-9 (MMP-9) knockout (-/-), heterozygote cystathionine beta synthase (CBS-/+), and endothelial nitric oxide synthase knockout (eNOS-/-) mouse brains were isolated. The MVEC were incorporated into collagen (3.2 mg/ml) gels and the decrease in collagen gel diameter at 24 h was used as an index of constrictive MVEC remodeling. Gels in the absence or presence of Hcy were incubated with muscimol or baclofen, a GABA-B receptor agonist. The results suggested that Hcy-mediated MVEC collagen gel constriction was ameliorated by muscimol, baclofen, MMP-9, and eNOS gene ablations. There was no effect of anti-alpha 3 integrin. However, Hcy-mediated brain MVEC collagen constriction was abrogated with anti-beta-1 integrin. The co-incubation of Hcy with L-arginine ameliorated the Hcy-mediated collagen gel constriction. The results of this study indicated amelioration of Hcy-induced MVEC collagen gel constriction by induction of nitric oxide through GABA-A and -B receptors.     

Expression of RANKL/OPG during bone remodeling in vivo

The interaction between receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation.Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen α1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The expression of bone formation markers was activated in the bone formation phase, followed by the stimulation of RANKL/OPG expression in the bone resorption phase, which confirmed that these molecules are key factors linking bone formation to resorption during bone remodeling.     

Mitochondrial remodeling in hepatic differentiation and dedifferentiation

Mitochondrial biogenesis and metabolism have recently emerged as important actors of stemness and differentiation. On the one hand, the differentiation of stem cells is associated with an induction of mitochondrial biogenesis and a shift from glycolysis toward oxidative phosphorylations (OXPHOS). In addition, interfering with mitochondrial biogenesis or function impacts stem cell differentiation. On the other hand, some inverse changes in mitochondrial abundance and function are observed during the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Yet although great promises in cell therapy might generate better knowledge of the mechanisms regulating the stemness and differentiation of somatic stem cells (SSCs)—which are preferred over embryonic stem cells (ESCs) and iPSCs because of ethical and safety considerations—little interest was given to the study of their mitochondria. This study provides a detailed characterization of the mitochondrial biogenesis occurring during the hepatogenic differentiation of bone marrow-mesenchymal stem cells (BM-MSCs). During the hepatogenic differentiation of BM-MSCs, an increased abundance of mitochondrial DNA (mtDNA) is observed, as well as an increased expression of several mitochondrial proteins and biogenesis regulators, concomitant with increased OXPHOS activity, capacity, and efficiency. In addition, opposite changes in mitochondrial morphology and in the abundance of several OXPHOS subunits were found during the spontaneous dedifferentiation of primary hepatocytes. These data support reverse mitochondrial changes in a different context from genetically-engineered reprogramming. They argue in favor of a mitochondrial involvement in hepatic differentiation and dedifferentiation.     

Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone

White adipose tissue is an important endocrine organ involved in the control of whole-body metabolism, insulin sensitivity, and food intake. To better understand these functions, 3T3-L1 cell differentiation was studied by using combined proteomic and genomic strategies. The proteomics approach developed here exploits velocity gradient centrifugation as an alternative to isoelectric focusing for protein separation in the first dimension. A 20- to 30-fold increase in the concentration of numerous mitochondrial proteins was observed during adipogenesis, as determined by mass spectrometry and database correlation analysis. Light and electron microscopy confirmed a large increase in the number of mitochondrion profiles with differentiation. Furthermore, mRNA profiles obtained by using Affymetrix GeneChips revealed statistically significant increases in the expression of many nucleus-encoded mitochondrial genes during adipogenesis. Qualitative changes in mitochondrial composition also occur during adipose differentiation, as exemplified by increases in expression of proteins involved in fatty acid metabolism and of mitochondrial chaperones. Furthermore, the insulin sensitizer rosiglitazone caused striking changes in mitochondrial shape and expression of selective mitochondrial proteins. Thus, although mitochondrial biogenesis has classically been associated with brown adipocyte differentiation and thermogenesis, our results reveal that mitochondrial biogenesis and remodeling are inherent to adipose differentiation per se and are influenced by the actions of insulin sensitizers.     

Computational analysis of bone remodeling during an anterior cervical fusion

The anterior cervical fusion is an established surgical procedure for spine stabilization after the removal of an intervertebral disc. However, it is not yet clear which bone graft represents the best choice and whether surgical devices can be efficient and beneficial for fusion. The aim of this work is to study the influence of the spine instrumentation on bone remodeling after a cervical spine surgery and, consequently, on the fusion process. A finite element model of the cervical spine was developed, having computed tomography images as input. Bone was modeled as a porous material characterized by the relative density at each point and the bone remodeling law was derived assuming that bone self-adapts in order to achieve the stiffest structure for the supported loads, with the total bone mass regulated by the metabolic cost of maintaining bone tissue. Apart from the analysis of healthy cervical spine, different surgical scenarios were tested: bone graft with or without a cage and the use of a stabilization plate system. Results showed that the anterior and posterior regions of the disc space are more important to stress transmission and that spinal devices reduce bone growth within bone grafts, being plate systems the most interfering elements. The material of the interbody cages plays a major role in fusion and, therefore, it should be carefully chosen.     

Plant chromatin -- epigenetics linked to ATP-dependent remodeling and architectural proteins

Studies in organisms belonging to different eukaryotic kingdoms have revealed that the structural state of chromatin is controlled by interactions of DNA, small RNAs and specific proteins, linked to a self-reinforcing complex network of biochemical activities involving histone and DNA modifications and ATP-dependent nucleosome remodeling. However, these findings must now be reinterpreted in light of the recent discovery of the highly dynamic character of interphase chromosomes exemplified by the constant flux of enzymatic and structural proteins through both eu- and heterochromatin and by short- and long-range chromosome movements in the nucleus. The available data on chromosome organization in Arabidopsis thaliana and links between proteins influencing chromatin structure and DNA and histone modifications documented in this model plant provide strong supportive evidence for the dynamic nature of chromosomes.     

Mitochondrial ROS in myocardial ischemia reperfusion and remodeling

Despite major progress in interventional and medical treatments, myocardial infarction (MI) and subsequent development of heart failure (HF) are still associated with high mortality. Both during ischemia reperfusion (IR) in the acute setting of MI, as well as in the chronic remodeling process following MI, oxidative stress substantially contributes to cardiac damage. Reactive oxygen species (ROS) generated within mitochondria are particular drivers of mechanisms contributing to IR injury, including induction of mitochondrial permeability transition or oxidative damage of intramitochondrial structures and molecules. But even beyond the acute setting, mechanisms like inflammatory signaling, extracellular remodeling, or pro-apoptotic signaling that contribute to post-infarction remodeling are regulated by mitochondrial ROS. In the current review, we discuss both sources and consequences of mitochondrial ROS during IR and in the chronic setting following MI, thereby emphasizing the potential therapeutic value of attenuating mitochondrial ROS to improve outcome and prognosis for patients suffering MI.     

Computational simulation of simultaneous cortical and trabecular bone change in human proximal femur during bone remodeling

In this study, we developed a numerical framework that computationally determines simultaneous and interactive structural changes of cortical and trabecular bone types during bone remodeling, and we investigated the structural correlation between the two bone types in human proximal femur. We implemented a surface remodeling technique that performs bone remodeling in the exterior layer of the cortical bone while keeping its interior area unchanged. A micro-finite element (μFE) model was constructed that represents the entire cortical bone and full trabecular architecture in human proximal femur. This study simulated and compared the bone adaptation processes of two different structures: (1) femoral bone that has normal cortical bone shape and (2) perturbed femoral bone that has an artificial bone lump in the inferomedial cortex. Using the proposed numerical method in conjunction with design space optimization, we successfully obtained numerical results that resemble actual human proximal femur. The results revealed that actual cortical bone, as well as the trabecular bone, in human proximal femur has structurally optimal shapes, and it was also shown that a bone abnormality that has little contribution to bone structural integrity tends to disappear. This study also quantitatively determined the structural contribution of each bone: when the trabecular adaptation was complete, the trabecular bone supported 54% of the total load in the human proximal femur while the cortical bone carried 46%.     

Extracellular matrix networks in bone remodeling

Bones are constantly remodeled throughout life to maintain robust structure and function. Dysfunctional remodeling can result in pathological conditions such as osteoporosis (bone loss) or osteosclerosis (bone gain). Bone contains 100s of extracellular matrix (ECM) proteins and the ECM of the various bone tissue compartments plays essential roles directing the remodeling of bone through the coupled activity of osteoclasts (which resorb bone) and osteoblasts (which produce new bone). One important role for the ECM is to serve as a scaffold upon which mineral is deposited. This scaffold is primarily type I collagen, but other ECM components are involved in binding of mineral components. In addition to providing a mineral scaffolding role, the ECM components provide structural flexibility for a tissue that would otherwise be overly rigid. Although primarily secreted by osteoblast-lineage cells, the ECM regulates cells of both the osteoblast-lineage (such as progenitors, mature osteoblasts, and osteocytes) and osteoclast-lineage (including precursors and mature osteoclasts), and it also influences the cross-talk that occurs between these two oppositional cells. ECM influences the differentiation process of mesenchymal stem cells to become osteoblasts by both direct cell-ECM interactions as well as by modulating growth factor activity. Similarly, the ECM can influence the development of osteoclasts from undifferentiated macrophage precursor cells, and influence osteoclast function through direct osteoclast cell binding to matrix components. This comprehensive review will focus on how networks of ECM proteins function to regulate osteoclast- and osteoblast-mediated bone remodeling. The clinical significance of these networks on normal bone and as they relate to pathologies of bone mass and geometry will be considered. A better understanding of the dynamic role of ECM networks in regulating tissue function and cell behavior is essential for the development of new treatment approaches for bone loss.     

Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.     

A molecular analysis of matrix remodeling and angiogenesis during long bone development

The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a ‘molecular map’ of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.