The role of the mitochondrial trans-sulfuration in cerebro-cardio renal dysfunction during trisomy down syndrome.

One in 700 children is born with the down syndrome (DS). In DS, there is an extra copy of X chromosome 21 (trisomy). Interestingly, the chromosome 21 also contains an extra copy of the cystathionine beta synthase (CBS) gene. The CBS activity is known to contribute in mitochondrial sulfur metabolism via trans-sulfuration pathway. We hypothesize that due to an extra copy of the CBS gene there is hyper trans-sulfuration in DS. We believe that understanding the mechanism of hyper trans-sulfuration during DS will be important in improving the quality of DS patients and towards developing new treatment strategies. We know that folic acid "1-carbon" metabolism (FOCM) cycle transfers the "1-carbon" methyl group to DNA (H3K4) via conversion of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) by DNMTs (the gene writers). The demethylation reaction is carried out by ten-eleven translocation methylcytosine dioxygenases (TETs; the gene erasers) through epigenetics thus turning the genes off/on and opening the chromatin by altering the acetylation/HDAC ratio. The S-adenosyl homocysteine hydrolase (SAHH) hydrolyzes SAH to homocysteine (Hcy) and adenosine. The Hcy is converted to cystathionine, cysteine and hydrogen sulfide (H2S) via CBS/cystathioneγ lyase (CSE)/3-mercaptopyruvate sulfurtransferase (3MST) pathways. Adenosine by deaminase is converted to inosine and then to uric acid. All these molecules remain high in DS patients. H2S is a potent inhibitor of mitochondrial complexes I-IV, and regulated by UCP1. Therefore, decreased UCP1 levels and ATP production can ensue in DS subjects. Interestingly, children born with DS show elevated levels of CBS/CSE/3MST/Superoxide dismutase (SOD)/cystathionine/cysteine/H2S. We opine that increased levels of epigenetic gene writers (DNMTs) and decreased in gene erasers (TETs) activity cause folic acid exhaustion, leading to an increase in trans-sulphuration by CBS/CSE/3MST/SOD pathways. Thus, it is important to determine whether SIRT3 (inhibitor of HDAC3) can decrease the trans-sulfuration activity in DS patients. Since there is an increase in H3K4 and HDAC3 via epigenetics in DS, we propose that sirtuin-3 (Sirt3) may decrease H3K4 and HDAC3 and hence may be able to decrease the trans-sulfuration in DS. It would be worth to determine whether the lactobacillus, a folic acid producing probiotic, mitigates hyper-trans-sulphuration pathway in DS subjects. Further, as we know that in DS patients the folic acid is exhausted due to increase in CBS, Hcy and re-methylation. In this context, we suggest that folic acid producing probiotics such as lactobacillus might be able to improve re-methylation process and hence may help decrease the trans-sulfuration pathway in the DS patients.

Technological advancements in jaggery-making processes and emission reduction potential via clean combustion for sustainable jaggery production: An overview.

Jaggery is a kind of unrefined non-centrifugal sugar (NCS) used mainly in Asia, Africa, Latin America, and the Caribbean. Traditionally, jaggery is produced by concentrating sugarcane juice in open pans with the help of bagasse combustion. However, due to thermal energy loss with flue gases and an unscientific approach in plant construction, jaggery plants have a poor thermal efficiency of less than 25%, poor emission characteristics, and a high bagasse consumption rate. Advanced jaggery-making techniques use solar energy and heat pumps for jaggery production. However, these techniques are in the early stage of development, and the literature indicates that these techniques should be used in conjuction with traditional ones to improve the performance of jaggery making plants. This literature review describes advances in jaggery-making methods, critically analyzed them, and provides a qualitative comparison of these methods. Further, gaps in the existing literature are identified and reported for future research direction. In addition, efforts have been made to quantify and estimate the emissions reduction and bagasse consumption potentials from the traditional jaggery industry to make this rural industry a sustainable and profitable business for rural entrepreneurs. The comparison with the recently developed clean combustion device exhibits that the harmful emissions from the jaggery industry could be reduced drastically viz. 95%-98% of PM2.5; 92%-95% of CO, and 52-60% of CO2, while saving more than 35% of bagasse consumption. Implemented at a national scale, it may reduce nearly 3% of all harmful emissions in the country, which is equally applicable elsewhere.

An evaluation of the safety of continuing trastuzumab despite overt left ventricular dysfunction.

Background: The major limitation in the use of trastuzumab therapy is cardiotoxicity. We evaluated the safety of a strategy of continuing trastuzumab in patients with breast cancer despite mild, asymptomatic left ventricular impairment. Methods: Charts of consecutive patients referred to a cardio-oncology clinic from January 2015 to March 2017 for decline in left ventricular ejection fraction (lvef), defined as a fall of 10 percentage points or more, or a value of less than 50% during trastuzumab therapy, were reviewed. The primary outcome of interest was change in lvef, measured before and during trastuzumab exposure and up to 3 times after initiation of cardiac medications during a median of 9 months. Results: All 18 patients referred for decline in lvef chose to remain on trastuzumab and were included. All patients were treated with angiotensin converting-enzyme inhibitors or beta-blockers, or both. After initiation of cardiac medications, lvef increased over time by 4.6 percentage points (95% confidence interval: 1.9 percentage points to 7.4 percentage points), approaching baseline values. Of the 18 patients, 17 (94%) were asymptomatic at all future visits. No deaths occurred in the group. Conclusions: Many patients with mildly reduced lvef and minimal heart failure symptoms might be able to continue trastuzumab without further decline in lvef, adverse cardiac events, or death when treated under the supervision of a cardiologist with close follow-up.

Clinical practice guidelines in breast cancer.

Background: A number of clinical practice guidelines (cpgs) concerning breast cancer (bca) screening and management are available. Here, we review the strengths and weaknesses of cpgs from various professional organizations and consensus groups with respect to their methodologic quality, recommendations, and implementability. Methods: Guidelines from four groups were reviewed with respect to two clinical scenarios: adjuvant ovarian function suppression (ofs) in premenopausal women with early-stage estrogen receptor-positive bca, and use of sentinel lymph node biopsy (slnb) after neoadjuvant chemotherapy (nac) for locally advanced bca. Guidelines from the American Society of Clinical Oncology (asco); Cancer Care Ontario's Program in Evidence Based Care (cco's pebc); the U.S. National Comprehensive Cancer Network (nccn); and the St. Gallen International Breast Cancer Consensus Conference were reviewed by two independent assessors. Guideline methodology and applicability were evaluated using the agree ii tool. Results: The quality of the cpgs was greatest for the guidelines developed by asco and cco's pebc. The nccn and St. Gallen guidelines were found to have lower scores for methodologic rigour. All guidelines scored poorly for applicability. The recommendations for ofs were similar in three guidelines. Recommendations by the various organizations for the use of slnb after nac were contradictory. Conclusions: Our review demonstrated that cpgs can be heterogeneous in methodologic quality. Low-quality cpg implementation strategies contribute to low uptake of, and adherence to, bca cpgs. Further research examining the barriers to recommendations-such as intrinsic guideline characteristics and the needs of end users-is required. The use of bca cpgs can improve the knowledge-to-practice gap and patient outcomes.

Potential Contribution of Antioxidant Mechanism in the Defensive Effect of Lycopene Against Partial Sciatic Nerve Ligation Induced Behavioral, Biochemical and Histopathological Modification in Wistar Rats.

Neuropathic pain is a severe and unbearable condition which arises due to activation of peripheral nociceptors after tissue damage, neuropathic pain is caused from anomalous physiology of central or peripheral nervous system and it may not be related to the ongoing tissue damage or inflammation. Involvement of oxidative damage has been reported in the pathophysiology of neuropathic pain. The purpose of this study was to examine the effect of lycopene to quench the free radicals produced as a result of the increased oxidative stress in neuropathic pain. Neuropathic pain was induced in wistar rats by partial sciatic nerve ligation. The effect was evaluated by assessing various behavioral parameters (thermal hyperalgesia, cold hyperalgesia), biochemical parameters (lipid peroxidation, reduced glutathione, superoxide dismutase and catalase) as well as histopathological parameters in sciatic nerve. During the experiment group of 8 rats each was administered drugs once daily intraperitonealy (I.P.) and naïve groups, sham group and sciatic nerve ligated group were treated with vehicle for the duration of 14 days. Partial sciatic nerve ligation (PSNL) significantly caused thermal hyperalgesia, cold hyperalgesia and oxidative damage compared to normal and sham groups. Daily administration of lycopene (25 mg/kg, 50 mg/kg) and gabapentin (100 mg/kg) considerably reversed hyperalgesia, cold hyperalgesia and attenuated oxidative stress when compared to control group. There was significant histological improvement in the in the architecture of myelinated and unmyelinated fibers. The results indicated that free radical generation mechanism might be involved in PSNL induced behavior, biochemical and histopathological changes in wistar rats.

Homocysteine Induced Cerebrovascular Dysfunction: A Link to Alzheimer's Disease Etiology.

A high serum level of homocysteine, known as hyperhomocystenemia (HHcy) is associated with vascular dysfunction such as altered angiogenesis and increased membrane permeability. Epidemiological studies have found associations between HHcy and Alzheimer's disease (AD) progression that eventually leads to vascular dementia (VaD). VaD is the second most common cause of dementia in people older than 65, the first being AD. VaD affects the quality of life for those suffering by drastically decreasing their cognitive function. VaD, a cerebrovascular disease, generally occurs due to cerebral ischemic events from either decreased perfusion or hemorrhagic lesions. HHcy is associated with the hallmarks of dementia such as tau phosphorylation, Aß aggregation, neurofibrillary tangle (NFT) formation, neuroinflammation, and neurodegeneration. Previous reports also suggest HHcy may promote AD like pathology by more than one mechanism, including cerebral microangiopathy, endothelial dysfunction, oxidative stress, neurotoxicity and apoptosis. Despite the corelations presented above, the question still exists - does homocysteine have a causal connection to AD? In this review, we highlight the role of HHcy in relation to AD by discussing its neurovascular effects and amelioration with dietary supplements. Moreover, we consider the studies using animal models to unravel the connection of Hcy to AD.

Interleukin-8 is a key mediator of FKBP51-induced melanoma growth, angiogenesis and metastasis.

BACKGROUND: FKBP51 is overexpressed in melanoma and impacts tumour cell properties. However, its comprehensive role in melanoma pathogenesis and underlying mechanism(s) remain elusive. METHODS: FKBP51 was stably silenced in aggressive melanoma cell lines and its effect examined in vitro and in mouse model. Histological/immunohistochemical analyses were performed to confirm metastasis, angiogenesis and neutrophil infiltration. Gene expression was analyzed by qRT-PCR, immunoblot and/or ELISA. NF-κB transcriptional activity and promoter binding were monitored by luciferase-based promoter-reporter and ChIP assays, respectively. Interleukin (IL)-8 inhibition was achieved by gene silencing or neutralising-antibody treatment. RESULTS: FKBP51 silencing reduced melanoma growth, metastasis, angiogenesis and neutrophil infiltration and led to IL-8 downregulation through NF-κB suppression in cell lines and tumour xenografts. IL-8 inhibition drastically decreased growth, migration and invasiveness of FKPB51-overexpressing cells; whereas its treatment partially restored the suppressed phenotypes of FKBP51-silenced melanoma cells. Interleukin-8 depletion in conditioned medium (CM) of FKBP51-overexpressing melanoma cells inhibited endothelial cell proliferation and capillary-like structure formation, whereas its treatment promoted these effects in endothelial cells cultured in CM of FKBP51-silenced melanoma cells. CONCLUSIONS: FKBP51 promotes melanoma growth, metastasis and angiogenesis, and IL-8 plays a key role in these processes. Thus, targeting of FKBP51 or its upstream or downstream regulatory pathways could lead to effective therapeutic strategies against melanoma.

Curcumin-primed exosomes mitigate endothelial cell dysfunction during hyperhomocysteinemia.

AIM: Exosomes, the nano-units (<200 nm), released from diverse cell types in the extracellular body fluid, possess non-immunogenic property and ability to cross the blood-brain barrier (BBB). Since exosomes carry biological information from their cells of origin, we hypothesize that priming cells with potential therapeutic agents release improved cellular contents through exosomes. Curcumin possesses anti-oxidative and anti-inflammatory properties and provides a promising treatment for cerebral diseases and therefore, the aim of the study is to establish that mouse brain endothelial cells (MBECs) when primed with curcumin (7.5 µM), release an alleviated exosome population that can help recover the endothelial cell (EC) layer permeability. MAIN METHODS: Homocysteine is a well-known causative factor of BBB disruption; therefore, homocysteine-treated ECs were used as a model of BBB disruption and curcumin-primed exosomes were utilized to check their potential for mitigating EC disruption. MBECs were treated with curcumin and exosomes were isolated by using ultracentrifugation and immunoprecipitation. Expression levels of junction proteins were detected by Western blot and immunocytochemistry assays. Endothelial cell permeability was analyzed with Fluorescein isothiocyanate-Bovine serum albumin (FITC-BSA) leakage assay using transwell permeable supports. KEY FINDINGS: Exosomes derived from curcumin-treated (primed) cells (CUR-EXO) alleviated oxidative stress, tight junctions (ZO-1, claudin-5, occludin), adherent junction (VE-cadherin) proteins and EC layer permeability induced during EC damage due to high homocysteine levels (hyperhomocysteinemia). SIGNIFICANCE: In conclusion, the study potentiates the use of CUR-EXO for cerebral diseases where drug delivery is still a challenge. The results also pave the way to novel translational therapies for cerebral diseases by maintaining and establishing therapeutic conservatories via primed exosomes.

Efficacy of Liposomal Monensin on the Enhancement of the Antitumour Activity of Liposomal Ricin in Human Epidermoid Carcinoma (KB) Cells.

The monensin, known to enhance the cytotoxicity of ricin and ricin-based immunotoxins is a very hydrophobic molecule and this limits its administration in optimum doses under in vivo conditions. In order to realise its full potential, monensin was intercalated into various liposomal formulations and its ability to potentiate the cytotoxicity of ricin liposomes in human epidermoid carcinoma (KB) cells was studied. It was observed that ricin cytotoxicity enhancing ability of monensin liposome depends on the surface charge as well as density and chain length of distearoyl phosphatidylethanolamine-methoxy polyethylene glycol present on the surface of liposomal monensin. Maximum potentiation on the cytotoxicity of liposomal ricin was observed by monensin entrapped in neutral liposome (106.5 fold) followed by negatively charged (94.2 fold) and positively charged liposome (90 fold). Studies on the effect of variation of density and chain length of distearoyl phosphatidylethanolamine-methoxy polyethylene glycol showed that neutral monensin liposomes having 2.5 mol% distearoyl phosphatidylethanolamine-methoxy polyethylene glycol with chain length of 2000 exhibits maximum potentiation (117.6 fold) on the cytotoxicity of ricin liposomes when the cellular uptake of monensin liposome was maximum (42.0%) and the zeta potential value on the surface of liposomes was -0.645. The present study has clearly shown that liposomal monensin is very effective in enhancing the cytotoxicity of liposomal ricin in human cancer cells and liposome can be used as in vivo deliver vehicle for monensin to potentiate the cytotoxicity of liposomal ricin to eliminate cancer cells.

Hydrogen sulfide attenuates neurodegeneration and neurovascular dysfunction induced by intracerebral-administered homocysteine in mice.

High levels of homocysteine (Hcy), known as hyperhomocysteinemia are associated with neurovascular diseases. H2S, a metabolite of Hcy, has potent anti-oxidant and anti-inflammatory activities; however, the effect of H2S has not been explored in Hcy (IC)-induced neurodegeneration and neurovascular dysfunction in mice. Therefore, the present study was designed to explore the neuroprotective role of H2S on Hcy-induced neurodegeneration and neurovascular dysfunction. To test this hypothesis we employed wild-type (WT) males ages 8-10 weeks, WT+artificial cerebrospinal fluid (aCSF), WT+Hcy (0.5 µmol/µl) intracerebral injection (IC, one time only prior to NaHS treatment), WT+Hcy+NaHS (sodium hydrogen sulfide, precursor of H2S, 30 µmol/kg, body weight). NaHS was injected i.p. once daily for the period of 7 days after the Hcy (IC) injection. Hcy treatment significantly increased malondialdehyde, nitrite level, acetylcholinestrase activity, tumor necrosis factor-alpha, interleukin-1 beta, glial fibrillary acidic protein, inducible nitric oxide synthase, endothelial nitric oxide synthase and decreased glutathione level indicating oxidative-nitrosative stress and neuroinflammation as compared to control and aCSF-treated groups. Further, increased expression of neuron-specific enolase, S100B and decreased expression of (post-synaptic density-95, synaptosome-associated protein-97) synaptic protein indicated neurodegeneration. Brain sections of Hcy-treated mice showed damage in the cortical area and periventricular cells. Terminal deoxynucleotidyl transferase-mediated, dUTP nick-end labeling-positive cells and Fluro Jade-C staining indicated apoptosis and neurodegeneration. The increased expression of matrix metalloproteinase (MMP) MMP9, MMP2 and decreased expression of tissue inhibitor of metalloproteinase (TIMP) TIMP-1, TIMP-2, tight junction proteins (zonula occulden 1) in Hcy-treated group indicate neurovascular remodeling. Interestingly, NaHS treatment significantly attenuated Hcy-induced oxidative stress, memory deficit, neurodegeneration, neuroinflammation and cerebrovascular remodeling. The results indicate that H2S is effective in providing protection against neurodegeneration and neurovascular dysfunction.

Production of extracellular polymeric substances (EPS) by Serratia sp.1 using wastewater sludge as raw material and flocculation activity of the EPS produced.

Growth profile and extracellular polymeric substances (EPS) production of Serratia sp.1 was studied in shake flask fermentation for 72 h using wastewater sludge as raw material. Maximum cell concentration of 6.7 × 10(9) cfu/mL was obtained at 48 h fermentation time. EPS dry weight, flocculation activity and dewaterability of different EPS (tightly bound or TB-EPS, loosely bound or LB-EPS and broth-EPS or B-EPS) were also measured. The highest concentration of LB-EPS (2.45 g/L) and TB-EPS (0.99 g/L) were attained at 48 h of fermentation. Maximum flocculation activity and dewaterability (ΔCST) of TB-EPS (76.4%, 14.5s and 76.5%, 15.5s), LB-EPS (67.8%, 8.1s and 64.7%, 7.6s) and broth EPS (61%, 6.1s and 70.4%, 6.8s) were obtained at 36 and 48 h of growth. Higher flocculation activity and dewaterability were achieved with TB-EPS than with the two other EPS. Characterization of TB-EPS and LB-EPS was done in terms of their protein and carbohydrate content. Protein content was much higher in TB-EPS where as carbohydrate content was only slightly higher in TB-EPS than LB-EPS. Morphology of the Serratia strain after fermentation in sludge and TSB was observed under a scanning electron microscope and the cell size was found to be bigger in the sludge medium than the TSB medium.

Repertoire of Protein Kinases Encoded in the Genome of Takifugu rubripes.

Takifugu rubripes is teleost fish widely used in comparative genomics to understand the human system better due to its similarities both in number of genes and structure of genes. In this work we survey the fugu genome, and, using sensitive computational approaches, we identify the repertoire of putative protein kinases and classify them into groups and subfamilies. The fugu genome encodes 519 protein kinase-like sequences and this number of putative protein kinases is comparable closely to that of human. However, in spite of its similarities to human kinases at the group level, there are differences at the subfamily level as noted in the case of KIS and DYRK subfamilies which contribute to differences which are specific to the adaptation of the organism. Also, certain unique domain combination of galectin domain and YkA domain suggests alternate mechanisms for immune response and binding to lipoproteins. Lastly, an overall similarity with the MAPK pathway of humans suggests its importance to understand signaling mechanisms in humans. Overall the fugu serves as a good model organism to understand roles of human kinases as far as kinases such as LRRK and IRAK and their associated pathways are concerned.

SU-E-T-622: A Rapid Hybrid VMAT-IMRT Planning Method Using an Abbreviated Beam Angle Optimization Search.

PURPOSE: To generate hybrid VMAT-IMRT treatment plans by utilizing an IMRT beam-angle-optimality (BAO) search under a commercially available TPS without the aid of custom optimization software or high performance computing. METHODS: The high dose modulation provided by IMRT beams can be exploited to improve the quality of VMAT treatments. To achieve this, a VMAT treatment was created where the gantry pauses at predetermined angles to deliver IMRT segments. To determine IMRT BAO, an IMRT plan ('poly-IMRT') was made with many beams (>30) equally spaced around the patient. For practical reasons and to reduce the total time to approximately 1 hour, BAO was approximated by removing one beam out of the set and noting the new objective score. Determining this'score penalty' for each of the beams serves as a proxy for true BAO. The hybrid plan was created by combining the VMAT arc with a user-determined number of top-ranked beams from the poly-IMRT set. The BAO from this approach was compared with a more rigorous method ('VMAT+1'), in which a VMAT plan was optimized with 1 IMRT beam at various angles, allowing a direct determination of objective score versus gantry angle. The overall hybrid planning process was demonstrated by creating separate plans for a SBRT lung patient, with dose normalized to the limiting maximum aorta dose. RESULTS: Large score penalties from poly-IMRT coincided with large score benefits from VMAT+1, indicating both methods identified the same optimal beams. The VMAT, IMRT, and hybrid plans delivered the prescription dose to 84.3%, 85.6% and 87.7% of the PTV and had homogeneity indices of 1.38, 1.41, and 1.32 respectively. Normal tissue doses were within 0.5%. CONCLUSION: The presented method can create hybrid VMAT-IMRT plans which combine delivery efficiency with improved target coverage. The planning process takes about an hour using a standard TPS.

MO-D-BRB-08: BEST IN PHYSICS (THERAPY) - A Real Time Dose Monitoring and Dose Reconstruction Tool for Patient Specific VMAT QA and Delivery.

PURPOSE: To develop a real time dose monitoring and dose reconstruction tool to identify and quantify sources of errors during patient specific VMAT delivery and QAMethods: The VMAT delivery monitor tool called Linac Data Monitor (LDM) has been developed that connects to the linac in clinical mode and displays, records and compares real-time machine parameters to the planned parameters. A new quantity called integral error keeps a running total of leaf overshoot and undershoots errors in each leaf pair multiplied by leaf width and the amount of time during which error exists in MU delivery. Another tool reconstructs pinnacle format delivered plan based on the saved machine logfile and recalculates actual delivered dose in patient anatomy. Delivery characteristics of various standard and hypofractionation VMAT plans delivered on Elekta Axesse and Synergy linacs were quantified. RESULTS: The MLC and gantry errors for all the treatment sites were 0.00±0.59mm and 0.05±0.31°, indicating a good MLC gain calibration. Standard fractionation plans had a larger gantry error than hypofractionation plans due to frequent dose rate changes. On average the MLC errors were negligible but larger errors of 4-6 mm and 2.5° were seen when dose rate varied frequently. Large gantry errors occurred during the acceleration and deceleration process, and correlated well with MLC errors (p<0.0001). PTV mean, minimum, maximum dose discrepancy were 0.87±0.21%, 0.99±0.59% and 1.18±0.52%. The other OAR doses were within 2.5% except a few that showed up to 5.6% discrepancy in maximum dose. Realtime displayed normalized total positive integral error (normalized to the total MUs) correlated linearly with MLC and gantry errors (p<0.001) and dosimetric discrepancy (PTVmean: p<0.01; PTVmax: p<0.067 and PTVmax: p<0.046). CONCLUSIONS: Errors may exist during complex VMAT planning and delivery. LDM is capable of detecting and quantifying mechanical and dosimetric errors at various stages of planning and delivery.

Mesenteric vascular remodeling in hyperhomocysteinemia.

Remodeling by its very nature implies synthesis and degradation of extracellular matrix components (such as elastin, collagen, and connexins). Most of the vascular matrix metalloproteinase (MMP) are latent because of the presence of constitutive nitric oxide (NO). However, during oxidative stress peroxinitrite (ONOO-) activates the latent MMPs and instigates vascular remodeling. Interestingly, in mesenteric artery, homocysteine (Hcy) decreases the NO bio-availability, and folic acid (FA, an Hcy-lowering agent) mitigates the Hcy-mediated mesentery artery dysfunction. Dimethylarginine dimethylaminohydrolase-2 (DDAH-2) and endothelial nitric oxide synthase (eNOS) increases NO production. The hypothesis was that the Hcy decreased NO bio-availability, in part, activating MMP, decreasing elastin, DDAH-2, eNOS and increased vasomotor response by increasing connexin. To test this hypothesis,the authors used 12-week-old C57BJ/L6 wild type (WT) and hyperhomocysteinemic (HHcy)-cystathione beta synthase heterozygote knockout (CBS+/-) mice. Blood pressure measurements were made by radio-telemetry. WT and MMP-9 knockout mice were administered with Hcy (0.67 mg/ml in drinking water). Superior mesenteric artery and mesenteric arcade were analyzed with light and confocal microscopy. The protein expressions were measured by western blot analysis. The mRNA levels for MMP-9 were measured by RT-PCR. The data showed decreased DDAH-2 and eNOS expressions in mesentery in CBS-/+ mice compared with WT mice. Immuno-fluorescence and western blot results suggest increased MMP-9 and connexin-40 expression in mesenteric arcades of CBS-/+ mice compared with WT mice. The wall thickness of third-order mesenteric artery was increased in CBS-/+ mice compared to WT mice. Hcy treatment increased blood pressure in WT mice. Interestingly, in MMP-9 KO, Hcy did not increase blood pressure. These results may suggest that HHcy causes mesenteric artery remodeling and narrowing by activating MMP-9 and decreasing DDAH-2 and eNOS expressions, compromising the blood flow, instigating hypertension, and acute abdomen pain.

X-ray imaging of differential vascular density in MMP-9-/-, PAR-1-/+, hyperhomocysteinemic (CBS-/+) and diabetic (Ins2-/+) mice.

Although protease activated receptor-1 (PAR-1) and matrix metalloproteinase-9 (MMP-9) play significant role in vascular remodelling in hyperhomocysteinemia (HHcy due to cystathionine beta synthase deficiency, CBS-/+) and diabetes, mechanism remains nebulous. We hypothesized that differential vascular density and remodelling in different vascular beds in HHcy and diabetes were responsible for an adaptive metabolic homeostasis during the pathogenesis. To test this hypothesis, vascular density in mice lacking PAR-1, MMP-9, CBS and Insulin-2 gene mutant (Ins2-/+, Akita) was measured and compared with wild type (WT, C57BL/6J) mice. The vascular density was detected by x-ray angiography using KODAK 4000 MM image station, using barium sulphate as contrasting agent. The % vascular density in the hearts of WT, CBS-/+ (HHcy), MMP-9-/-, PAR-1-/+ and Ins2-/+ (type-1 diabetes) was 100 ± 2.8, 85 ± 3.3, 90 ± 3.3, 95 ± 3.8 and 73 ± 1.7, respectively. The vascular density in CBS-/+ and Akita hearts decreased while it was increased in lungs of CBS-/+ and MMP-9-/-.There was decreased vascular density in liver and kidney of Akita mice. Vascular density in brain, kidney and mesentery was decreased in CBS-/+ mice. These findings support the notation that metabolic derangement in diabetes and HHcy causes the chronic decline and/or rarefaction in vascular density.

Homocysteine enriched diet leads to prolonged QT interval and reduced left ventricular performance in telemetric monitored mice.

BACKGROUND AND AIMS: Homocysteine (Hcy) is a sulfur-containing, non-protein amino acid produced in the metabolic pathway of methionine. Hyperhomocysteinemia is associated with cerebro- and cardiovascular disease in industrialized countries, mostly resulting from protein rich diet and sedentary life style. Matrix metalloproteinases are involved in cardiac remodeling, leading to degradation of intercellular junctions, cardiac connexins and basement membranes. The study was designed to investigate the relationship between Hcy, cardiac remodeling, cardiac performance, and rhythm disturbances in an animal model of hyperhomocysteinemia. We tested the hypothesis that induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 leads to connexin 40, connexin 43, connexin 45 expression changes contributing to decreased cardiac performance and disturbed atrioventricular conduction. METHODS AND RESULTS: Hcy was added to drinking water of male C57/BL6J mice to achieve moderate Hcy blood levels. ECG was monitored in conscious mice with a telemetric ECG device; echocardiography was used for assessment of left ventricular function. Immunoblotting was used to evaluate matrix metalloproteinase-2, matrix metalloproteinase-9, connexin 40, connexin 43, and connexin 45 expression in cardiac tissue. Animals fed Hcy showed significant prolongation of QRS, QTc, and PR intervals along with reduced left ventricular function. Western blotting showed increased expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and decreased expression of connexin 40, 43, and 45. CONCLUSION: Hcy has been identified as a nutritional factor contributing to cardiovascular disease. Cardiac remodeling induced by matrix metalloproteinase-2 and matrix metalloproteinase-9 and decreased expression of connexin 40, 43, and 45 appears to play a role in the pathomechanism of atrioventricular conduction delay and ventricular dilatation in hyperhomocysteinemia.