Expedited Surgical Care of Appendicitis is Associated With Improved Resource Utilization.

INTRODUCTION: Appendectomy for acute appendicitis is the most common pediatric intra-abdominal operation. Current literature supports the notion that modest in-hospital, preoperative delays are not associated with greater patient morbidity. However, there is less certainty regarding the role that hour-of-presentation plays in determining the timing of surgery. Thus, we aimed to evaluate how after-hours presentation may relate to the timing of surgery and to assess the outcomes and resource utilization associated with expedited appendectomy compared to nonexpedited. METHODS: Patient records for children who underwent an appendectomy at a freestanding pediatric hospital from 2015 to 2019 were reviewed. Business hour presentations were defined as arrival at the emergency department from 7 AM to 6 PM. Primary outcomes were hospital length of stay (LOS), cost derived from the Pediatric Health Information System database, perforation, surgical complications, and 30-day readmissions. RESULTS: Nine hundred forty-two patients underwent appendectomy over the study period. The median time to OR was 2.0 h in the expedited cohort and 9.8 h in the nonexpedited group. Presentation during business hours was associated with 4.4 higher odds (P < 0.001) of expedited workflow. Expedited appendectomies were associated with shorter hospital LOS (11.5 h, P < 0.001), less costly admissions ($1,155, P < 0.001); LOS measured in midnights, perforation and readmission rates were similar between groups. CONCLUSIONS: We found reduced resource utilization associated with expedited appendectomy. Additionally, the demonstrated association between the time of presentation to the emergency department (ED) and the timing of surgery may be utilized to inform staffing and resource deployment decisions. Further research regarding the generalizability and sustainability of an expedited presurgical workflow in pediatric appendectomy is certainly indicated.

The costs and benefits of emergent surgical workflow for acute appendicitis in children.

BACKGROUND: Controversy exists regarding how operative timing affects patient safety and resource utilization for acute appendicitis. Over 3 years, our institution trialed efforts to optimize appendectomy workflow. Our aim is to describe the ramifications of expediting appendectomy and implementing standardized protocols relative to historic controls. METHODS: Patient records at a freestanding children's hospital were reviewed from synchronized 6-month periods from 2019 to 2021. During Year 1 (historic), no standardized workflows existed. In Year 2 (expedited), appendicitis management was protocoled using a clinical quality improvement bundle, which included performing appendectomies within two hours of diagnosis. In Year 3 (QI), operative timing was relaxed to the same calendar day while all prior QI initiatives continued. Descriptive statistics were performed, using hospital length of stay (LOS) as the primary outcome. RESULTS: 298 patients underwent appendectomy for acute appendicitis. The median expedited workflow LOS was 15.3 hours shorter (p = 0.003) than historic controls; however, this was sustained despite relaxation of surgical urgency in the QI workflow. No differences in perforation rates were observed. During the expedited workflow, OR overtime staffing expense increased by $90,000 with no significant change in hospital costs. In multivariate regression, perforation was the only variable associated with LOS. CONCLUSION: Hospital LOS can be shortened by expediting appendectomy. However, in our institution this did not decrease hospital costs and was furthermore balanced by higher personnel expenses. A sustained decrease in LOS after relaxing operative urgency standards implies that concurrent QI initiatives represent a more effective and cost-efficient strategy to decrease hospital resource utilization. LEVEL OF EVIDENCE: Level III.

Impact of the Affordable Care Act's Medicaid expansion on tertiary pediatric surgical care.

BACKGROUND: Many children gained insurance with the 2014 Affordable Care Act's (ACA) Medicaid Expansion (ME), yet its impact on access to pediatric tertiary surgical care remains unknown. We examined the effect of ME on rates of elective, ambulatory surgery (EAS), especially among publicly-insured and ethnoracial-minority patients. METHODS: Surgical patients ≤18 years between 2012 and 2018 were identified using the Pediatric Health Information System. Interrupted time series analyses were conducted to predict the monthly proportion of publicly-insured patients and EAS rates in ME and nonexpansion states. RESULTS: 3,270,842 patients were included. Nonexpansion states demonstrated a 1.10% (p<0.05) increase in the proportion of publicly-insured patients at ACA implementation, which then plateaued. No immediate change was observed in ME states, but there was an annual 1.08% (p<0.01) decrease in subsequent years. Publicly-insured EAS rates decreased by 1.09% (p<0.01) in nonexpansion states; no change was observed in ME states. A 3.36% (p<0.01) increase in EAS rates was observed in nonexpansion and ME states. The gap in EAS rates increased between private and publicly-insured patients in nonexpansion, but not ME states. CONCLUSIONS: Increased coverage for children in ME states was not associated with more access to tertiary pediatric surgical care; however, while nonexpansion states saw an increase in insurance-based disparities, ME states did not. Though insurance coverage is critical to access, other factors may be contributing to persistent disparities in access to pediatric surgical care.

Timeliness of pediatric surgical appendicitis care is associated with time of hospital admission.

BACKGROUND: Despite the frequency of acute appendicitis in children, there is no evidence-based consensus surrounding the urgency of the operation if a diagnosis is made after regular business hours. Although a modest delay in time to operation does not increase disease severity, postponing cases to the next calendar day may be associated with higher resource utilization. We aimed to evaluate the trend of delaying appendectomies to the next calendar day and its associated outcomes. METHODS: We queried the Pediatric Health Information System to analyze appendectomy patients younger than 18 y of age from 2010 to 2018. Same-day appendectomy and next-day appendectomy cohorts were created using admission hour and operative day. Healthcare cost, length of stay, surgical complications, and 30-day readmission rates were collected. Bivariate analyses and multivariable regressions were used to evaluate groups stratified by time of presentation. RESULTS: During the study period, 113,662 appendectomies were performed, comprising 88,715 (78.1%) same-day appendectomies and 24,947 (21.9%) next-day appendectomies. A higher proportion of same-day appendectomies (80.5%) were performed during hours 12:00am to 5:00pm and 19.5% were performed during hours 6:00pm to 11:00pm. The trend of next-day appendectomies increased during the study period from 13.9% to 20.2%. This was primarily evident in the 6:00pm to 11:00pm period. The 5:00pm cutoff was most predictive of a next-day appendectomy. Next-day appendectomies had similar rates of surgical complications; however, they were associated with higher costs, longer lengths of stay, and higher readmission rates. CONCLUSION: As the understanding of appendicitis urgency has changed, a more tempered approach of delivering surgical care has trended. Although short delays appear safe, postponement to the next calendar day is associated with higher resource utilization.

Trends in gastrostomy tube placement with concomitant Nissen fundoplication for infants and young children at Pediatric Tertiary Centers.

PURPOSE: In infants and toddlers, gastrostomy tube placement (GT) is typically accompanied by consideration of concomitant Nissen fundoplication (NF). Historically, rates of NF have varied across providers and institutions. This study examines practice variation and longitudinal trends in NF at pediatric tertiary centers. METHODS: Patients ≤ 2 years who underwent GT between 2008 and 2018 were identified in the Pediatric Health Information System database. Patient demographics and rates of NF were examined. Descriptive statistics were used to evaluate the variation in the proportion of GT with NF at each hospital, by volume and over time. RESULTS: 40,348 patients were identified across 40 hospitals. Most patients were male (53.8%), non-Hispanic white (49.5%) and publicly-insured (60.4%). Rates of NF by hospital varied significantly from 4.2 to 75.2% (p < 0.001), though were not associated with geographic region (p = 0.088). Rates of NF decreased from 42.8% in 2008 to 14.2% in 2018, with a mean annual rate of change of - 3.07% (95% CI - 3.53, - 2.61). This trend remained when stratifying hospitals into volume quartiles. CONCLUSION: There is significant practice variation in performing NF. Regardless of volume, the rate of NF is also decreasing. Objective NF outcome measurements are needed to standardize the management of long-term enteral access in this population.

A Rare Case of Contralateral Diaphragm Paralysis following Birth Injury with Brachial Plexus Palsy: A Case Report and Review of the Literature.

Clinical History. A 4.4 kg male was born to a 25-year-old, G2P1, nondiabetic woman at 39 and 5/7 weeks. Delivery was complicated by shoulder dystocia requiring forceps-assisted vaginal delivery, resulting in left arm Erb's palsy secondary to left brachial plexus injury. He was born with low muscle tone and bradycardia and subsequently required intubation for poor respiratory effort. He was extubated on day one of life but continued to be tachypneic and have borderline oxygen saturation, requiring intensive care. Chest radiographs demonstrated a progressive clearing of his lung fields, consistent with presumptively diagnosed meconium aspiration. However, a persistent elevation of the right hemidiaphragm was noted, and his tachypnea and increased work of breathing continued. Focused ultrasound of the diaphragm was performed, confirming decreased motion of the right hemidiaphragm. Following a multidisciplinary discussion, thoracoscopic right diaphragm plication was performed on the 33rd day of life. He was extubated postoperatively and subsequently weaned to room air with a notable decrease in tachypnea over 48 hours. He was discharged on postoperative day 12 and continues to thrive at 6 months of age without respiratory embarrassment. Purpose. Ipsilateral phrenic nerve injury with diaphragm paralysis from shoulder dystocia during vaginal delivery is a recognized phenomenon. Herein, we present a case of contralateral diaphragm paralysis in order to draw attention to the clinician that this discordance is possible. Key Points. According to Raimbault et al., clinical management of newborns who experience birth injury is a multidisciplinary effort. According to Fitting and Grassino, though most cases of phrenic nerve injuries are ipsilateral to shoulder dystocia brachial plexus palsy, contralateral occurrence is possible and should be considered. According to Waters, diaphragm plication is a safe and effective operation.

Quantifying postoperative sleep loss associated with increased pain in children undergoing a modified Nuss operation.

PURPOSE: The presence of pain may interrupt sleep and impede normal postoperative recovery; however, no prior studies have quantified sleep loss due to pain in children undergoing inpatient surgery. Wearable accelerometers objectively measure sleep patterns in children. We aimed to quantify sleep loss associated with patient reported pain scores after a Modified Nuss operation. METHODS: Ten patients undergoing Modified Nuss operations were recruited during their inpatient stay. Children wore an Actigraph GT3X-BT accelerometer postoperatively during their hospital stay. Hourly sleep minutes were recorded using the Actigraph between 10 pm and 6 am. Patient reported pain scores were abstracted from patient charts. Mixed linear regression models, adjusting for within-subject random effects, were estimated to quantify the association between hourly sleep minutes and patient reported pain scores. RESULTS: Patients were 30% female, with an average age of 15.7 years (range 13-22). The majority (70%) of patients were white non-Hispanic. All patients received a patient controlled analgesic pump. Average postoperative length of stay was 4.8 days (range 4.0-6.0; SD = 0.8). A total of 240 sleep hours and associated pain scores were analyzed. Patients slept on average 48 min per hour. Mixed model analysis predicted that a 1-point increase in pain score was associated with 2.5 min per hour less sleep time. CONCLUSION: Increases in patient-reported pain scores are associated with sleep loss after a Modified Nuss operation. Objectively quantifying sleep loss associated with postoperative pain using accelerometer data may help clinicians better understand their patient's level of pain control. Our findings provide the basis for future studies aimed at more accurately titrating pain medication to optimize sleep and speed up recovery. LEVEL OF EVIDENCE: Case Series Without Comparison Group, Level IV.

Diaphragmatic paralysis after phrenic nerve injury in newborns.

BACKGROUND: Phrenic nerve injury (PNI) from birth trauma is a recognized phenomenon, generally occurring with ipsilateral brachial plexus palsy (BPP). In severe cases, PNI results in diaphragm paresis (DP) and respiratory insufficiency. Surgical diaphragmatic plication (SDP) is a potential management strategy for patients with PNI and DP, but timing and outcomes associated with SDP have not been rigorously studied. METHODS: Records from 49 tertiary United States pediatric hospitals in the Pediatric Health Information System from 2004 to 2018 were analyzed. The study cohort included patients diagnosed with BPP from birth trauma who were documented to have PNI or DP. Patients who underwent congenital cardiac operations were excluded. RESULTS: A total of 5832 patients were identified with BPP from birth trauma during the study period, 122 (2%) of whom were found to have concomitant DP. Of those, 65 (53%) were male, 39 (32%) were infants of diabetic mothers, 80 (65%) required mechanical ventilation, and 33 (27%) underwent SDP. SDP was performed at a median (range) age of 36 (7-95) days. Median (range) total and postoperative hospital lengths of stay (LOS) were 34 (6-180) and 15 (4-132) days, respectively. There was also an observed increase in post-operative LOS with increase in age at operation. CONCLUSION: Neonatal DP is rare and is managed with SDP in a minority of instances. Age at repair affects total and postoperative length of stay, proxies for resource utilization and morbidity. Repair prior to 45 days of life appears to result in a shorter postoperative hospital stay. This analysis will help guide surgeons with respect to indications and operative timing for infant DP. TYPE OF STUDY: Retrospective Comparative Study. LEVEL OF EVIDENCE: Level III.

Trends in perioperative opioid and non-opioid utilization during ambulatory surgery in children.

BACKGROUND: In the midst of our national opioid crisis, recommendations have encouraged judicious stewardship of opioid prescription through the expanded use of non-opioid analgesic medications. This study aims to characterize trends in perioperative pain medication use for children undergoing ambulatory operations. METHODS: A cross-sectional, retrospective review was conducted using the Pediatric Health Information System. Patients younger than 18 years of age who underwent ambulatory surgery during 2010 to 2017 by one of five surgical subspecialties (otolaryngology, general pediatric, plastic or reconstructive, orthopedics, and urology) were included. Medications were identified using Current Procedural Terminology codes based on billing information for 18 commonly used analgesics along with the route of administration during their encounter. RESULTS: A total of 1,795,329 patients with a median age of 10 years were identified, of whom 84.3% received an opioid or non-opioid analgesic. Opioid use in the perioperative setting for ambulatory procedures decreased during the study period from 74.9% to 66.9% as a proportion of total analgesic prescriptions. Among opioids commonly used, intravenous morphine decreased the most from 19.8% to 15.4%, and intravenous hydromorphone and oral oxycodone use remained largely unchanged. Conversely, non-opiate medications increased, specifically intravenous ketorolac from 8.4% to 13.6%, and intravenous acetaminophen use increased from 0% to 8.5%. Intravenous acetaminophen use more than doubled between 2013 and 2017 (3.4% to 8.2%) and was accompanied by a decrease in oral acetaminophen use (14.4% to 9.3%). CONCLUSION: Overall, perioperative opioid utilization appears to be decreasing in favor of non-opioid analgesics. Other trends, such as increased intravenous acetaminophen, raise concerns for the cost effectiveness of perioperative analgesia and resource utilization.

Tetrahydrocurcumin ameliorates homocysteine-mediated mitochondrial remodeling in brain endothelial cells.

Homocysteine (Hcy) causes endothelial dysfunction by inducing oxidative stress in most neurodegenerative disorders. This dysfunction is highly correlated with mitochondrial dynamics such as fusion and fission. However, there are no strategies to prevent Hcy-induced mitochondrial remodeling. Tetrahydrocurcumin (THC) is an anti-inflammatory and anti-oxidant compound. We hypothesized that THC may ameliorates Hcy-induced mitochondria remodeling in mouse brain endothelial cells (bEnd3) cells. bEnd3 cells were exposed to Hcy treatment in the presence or absence of THC. Cell viability and autophagic cell death were measured with MTT and MDC staining assay. Reactive oxygen species (ROS) production was determined using DCFH-DA staining by confocal microscopy. Autophagy flux was assessed using a conventional GFP-microtubule-associated protein 1 light chain 3 (LC3) dot assay. Interaction of phagophore marker LC-3 with mitochondrial receptor NIX was observed by confocal imaging. Mitochondrial fusion and fission were evaluated by western blot and RT-PCR. Our results demonstrated that Hcy resulted in cell toxicity in a dose-dependent manner and supplementation of THC prevented the detrimental effects of Hcy on cell survival. Furthermore, Hcy also upregulated fission marker (DRP-1), fusion marker (Mfn2), and autophagy marker (LC-3). Finally, we observed that Hcy activated mitochondrial specific phagophore marker (LC-3) and co-localized with the mitochondrial receptor NIX, as viewed by confocal microscopy. Pretreatment of bEnd3 with THC (15 µM) ameliorated Hcy-induced oxidative damage, mitochondrial fission/fusion, and mitophagy. Our studies strongly suggest that THC has beneficial effects on mitochondrial remodeling and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy) induced mitochondrial dysfunction.

Inhibition of MMP-9 attenuates hypertensive cerebrovascular dysfunction in Dahl salt-sensitive rats.

Hypertensive cerebropathy is a pathological condition associated with cerebral edema and disruption of the blood-brain barrier. However, the molecular pathways leading to this condition remains obscure. We hypothesize that MMP-9 inhibition can help reducing blood pressure and endothelial disruption associated with hypertensive cerebropathy. Dahl salt-sensitive (Dahl/SS) and Lewis rats were fed with high-salt diet for 6 weeks and then treated without and with GM6001 (MMP inhibitor). Treatment of GM6001 (1.2 mg/kg body weight) was administered through intraperitoneal injections on alternate days for 4 weeks. GM6001 non-administered groups were given vehicle (0.9% NaCl in water) treatment as control. Blood pressure was measured by tail-cuff method. The brain tissues were analyzed for oxidative/nitrosative stress, vascular MMP-9 expression, and tight junction proteins (TJPs). GM6001 treatment significantly reduced mean blood pressure in Dahl/SS rats which was significantly higher in vehicle-treated Dahl/SS rats. MMP-9 expression and activity was also considerably reduced in GM6001-treated Dahl/SS rats, which was otherwise notably increased in vehicle-treated Dahl/SS rats. Similarly MMP-9 expression in cerebral vessels of GM6001-treated Dahl/SS rats was also alleviated, as devised by immunohistochemistry analysis. Oxidative/nitrosative stress was significantly higher in vehicle-treated Dahl/SS rats as determined by biochemical estimations of malondialdehyde, nitrite, reactive oxygen species, and glutathione levels. RT-PCR and immunohistochemistry analysis further confirmed considerable alterations of TJPs in hypertensive rats. Interestingly, GM6001 treatment significantly ameliorated oxidative/nitrosative stress and TJPs, which suggest restoration of vascular integrity in Dahl/SS rats. These findings determined that pharmacological inhibition of MMP-9 in hypertensive Dahl-SS rats attenuate high blood pressure and hypertension-associated cerebrovascular pathology.

Tetrahydrocurcumin ameliorates homocysteinylated cytochrome-c mediated autophagy in hyperhomocysteinemia mice after cerebral ischemia.

High levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy), contribute to autophagy and ischemia/reperfusion injury (I/R). Previous studies have shown that I/R injury and HHcy cause increased cerebrovascular permeability; however, the associated mechanism remains obscure. Interestingly, during HHcy, cytochome-c becomes homocysteinylated (Hcy-cyto-c). Cytochrome-c (cyto-c) transports electrons and facilitates bioenergetics in the system. However, its role in autophagy during ischemia/reperfusion injury is unclear. Tetrahydrocurcumin (THC) is a major herbal antioxidant and anti-inflammatory agent. Therefore, the objective of this study was to determine whether THC ameliorates autophagy during ischemia/reperfusion injury by reducing homocysteinylation of cyto-c in hyperhomocysteinemia pathological condition. To test this hypothesis, we employed 8-10-week-old male cystathionine-beta-synthase heterozygote knockout (CBS⁺/⁻) mice (genetically hyperhomocystemic mice). Experimental group was: CBS⁺/⁻, CBS⁺/⁻ + THC (25 mg/kg in 0.1% DMSO dose); CBS ⁺/⁻/I/R, and CBS⁺/⁻/I/R + THC (25 mg/kg in 0.1% DMSO dose). Ischemia was performed for 30 min and reperfusion for 72 h. THC was injected intra-peritoneally (I.P.) once daily for a period of 3 days after 30 min of ischemia. The infarct area was measured using 2,3,5-triphenyltetrazolium chloride staining. Permeability was determined by brain edema and Evans Blue extravasation. The brain tissues were analyzed for oxidative stress, matrix metalloproteinase-9 (MMP-9), damage-regulated autophagy modulator (DRAM), and microtubule-associated protein 1 light chain 3 (LC3) by Western blot. The mRNA levels of S-adenosyl-L-homocysteine hydrolases (SAHH) and methylenetetrahydrofolate reductase (MTHFR) genes were measured by quantitative real-time polymerase chain reaction. Co-immunoprecipitation was used to determine the homocysteinylation of cyto-c. We found that brain edema and Evans Blue leakage were reduced in I/R + THC-treated groups as compared to sham-operated groups along with reduced brain infarct size. THC also decreased oxidative damage and ameliorated the homocysteinylation of cyto-c in-part by MMP-9 activation which leads to autophagy in I/R groups as compared to sham-operated groups. This study suggests a potential therapeutic role of dietary THC in cerebral ischemia.

Mitochondrial mitophagic mechanisms of myocardial matrix metabolism and remodelling.

High levels of homocysteine (Hcy), known as hyperhomocysteinmia (HHcy), are correlated with an increase in extracellular matrix remodelling (ECM) via the matrix metalloproteinases (MMPs) and plasminogen/plasmin system. This results in an increase deposition of collagen that leads to endothelial-myocyte (EM) and myocyte-myocyte (MM) uncoupling; the physiological consequences are a plethora of cardiovascular pathologies. Homocysteine-induced increase in intracellular and mitochondrial Ca(2+) plays an important role in increasing reactive oxygen species (ROS) within mitochondria and instigating mitophagy within the cell. This occurs via several Hcy-mitigated processes: agonizing N-methyl-d-aspartate receptor-1 (NMDA-R1), decreasing expression of peroxisome proliferator activator receptor (PPAR) [thereby increasing oxidation], impairing Ca(2+) handling via Na(+)/Ca(2+) exchanger (NCX1) and Sarco endoplasmic reticulum Ca(2+) ATPase (SERCA-2a). The end result is an increase in ROS that directly or indirectly lead to MMP activation within mitochondria or the cytoplasm. Hcy induces a mitochondrial permeability transition that allows MMPs to be released from mitochondria thereby metabolizing matrix and impairing cardiac function. Further work remains to be elucidated concerning the specific mitochondrial mitophagic mechanisms under which matrix metabolism and remodelling occurs. Moreover, the therapeutic implications of NMDA and PPAR ligands are some promise to patient.

Autophagy and heart failure: a possible role for homocysteine.

Autophagy is a process used for intracellular digestion of organelles and proteins and has special relevance to the long-lived cardiomyocytes in heart disease. The pathway for autophagy and all its mediators remain to be elucidated, but involve such proteins as Atg, Beclin-1, LAMP-2, BH3, Bcl2, PI3K Kinase as well as a plethora of others. It is still not entirely clear whether autophagy is destructive or beneficial to the cell; evidence suggests that the answer is case-specific. For instance, autophagy appears to preserve cell life under cases of ischemia in I/R injury, but is detrimental during reperfusion. High levels of homocysteine (Hcy), a sulfur-containing amino acid, have been shown to be an independent risk factor for chronic heart failure. There are several links to induction and repression of autophagy and Hcy; the following connections to Hcy and autophagy have been made: intracellular nitrous oxide production, intracellular calcium production, and reactive oxygen species production. Further work remains to be elucidated concerning the specific mechanisms under which autophagy occurs and possible Hcy-mediated connections. Moreover, the therapeutic implications might be of some promise to patients.

Homocysteine mediated decrease in bone blood flow and remodeling: role of folic acid.

Deficiencies in folate lead to increased serum concentrations of homocysteine (Hcy), which is known as hyperhomocysteinemia (HHcy), is associated with bone disorders. Although, Hcy accumulates collagen in bone and contribute to decrease in bone strength. The mechanism of Hcy induced bone loss and remodeling is unclear. Therefore, the present study was aimed to determine the role of folic acid (FA) in genetically HHcy-associated decrease in bone blood flow and remodeling. Wild type (WT) and cystathionine-ß-synthase heterozygous (CBS+/-) mice were used in this study and supplemented with or without FA (300 mg/kg, Hcy reducing agent) in drinking water for 6 weeks. The tibial bone blood flow was measured by laser Doppler and ultrasonic flow probe method. The tibial bone density (BD) was assessed by dual energy X-ray absorptiometry. The bone homogenates were analyzed for oxidative stress, NOX-4 as oxidative marker and thioredoxin-1 (Trx-1) as anti-oxidant marker, bone remodeling (MMP-9) and bio-availability of nitric oxide (eNOS/iNOS/NO) by Western blot method. The results suggested that there was decrease in tibial blood flow in CBS+/- mice. The BD was also reduced in CBS+/- mice. There was an increase in NOX-4, iNOS, MMP-9 protein as well as MMP-9 activity in CBS+/- mice and decrease in Trx-1, eNOS protein levels, in part by decreasing NO bio-availability in CBS+/- mice. Interestingly, these effects were ameliorated by FA and suggested that FA supplementation may have therapeutic potential against genetically HHcy induced bone loss.

Hyperhomocysteinemia decreases bone blood flow.

Elevated plasma levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), are associated with osteoporosis. A decrease in bone blood flow is a potential cause of compromised bone mechanical properties. Therefore, we hypothesized that HHcy decreases bone blood flow and biomechanical properties. To test this hypothesis, male Sprague-Dawley rats were treated with Hcy (0.67 g/L) in drinking water for 8 weeks. Age-matched rats served as controls. At the end of the treatment period, the rats were anesthetized. Blood samples were collected from experimental or control rats. Biochemical turnover markers (body weight, Hcy, vitamin B(12), and folate) were measured. Systolic blood pressure was measured from the right carotid artery. Tibia blood flow was measured by laser Doppler flow probe. The results indicated that Hcy levels were significantly higher in the Hcy-treated group than in control rats, whereas vitamin B(12) levels were lower in the Hcy-treated group compared with control rats. There was no significant difference in folate concentration and blood pressure in Hcy-treated versus control rats. The tibial blood flow index of the control group was significantly higher (0.78 ± 0.09 flow unit) compared with the Hcy-treated group (0.51 ± 0.09). The tibial mass was 1.1 ± 0.1 g in the control group and 0.9 ± 0.1 in the Hcy-treated group. The tibia bone density was unchanged in Hcy-treated rats. These results suggest that Hcy causes a reduction in bone blood flow, which contributes to compromised bone biomechanical properties.

Hydrogen sulfide mitigates transition from compensatory hypertrophy to heart failure.

We reported previously that although there is disruption of coordinated cardiac hypertrophy and angiogenesis in transition to heart failure, matrix metalloproteinase (MMP)-9 induced antiangiogenic factors play a vital role in this process. Previous studies have shown the cardioprotective role of hydrogen sulfide (H2S) in various cardiac diseases, but its role during transition from compensatory hypertrophy to heart failure is yet to be unveiled. We hypothesize that H2S induces MMP-2 activation and inhibits MMP-9 activation, thus promoting angiogenesis, and mitigates transition from compensatory cardiac hypertrophy to heart failure. To verify this, aortic banding (AB) was created to mimic pressure overload in wild-type (WT) mice, which were treated with sodium hydrosulfide (NaHS, H2S donor) in drinking water and compared with untreated control mice. Mice were studied at 3 and 8 wk. In the NaHS-treated AB 8 wk group, the expression of MMP-2, CD31, and VEGF was increased while the expression of MMP-9, endostatin, angiostatin, and tissue inhibitor of matrix metalloproteinase (TIMP)-3 was decreased compared with untreated control mice. There was significant reduction in fibrosis in NaHS-treated groups. Echocardiograph and pressure-volume data revealed improvement of cardiac function in NaHS-treated groups over untreated controls. These results show that H2S by inducing MMP-2 promotes VEGF synthesis and angiogenesis while it suppresses MMP-9 and TIMP-3 levels, inhibits antiangiogenic factors, reduces intracardiac fibrosis, and mitigates transition from compensatory hypertrophy to heart failure.

Electrical stimulation of cardiomyocytes activates mitochondrial matrix metalloproteinase causing electrical remodeling.

Cardiac arrhythmias, instigated by mechanical and electrical remodeling, are associated with activation of extracellular matrix metalloproteinases (MMPs). However, the connection between intracellular MMPs activation and arrhythmogenesis is not well established. Previously, we determined localization of MMP in the mitochondria using confocal microscopy. We tested the hypothesis that electrical pacing induces the activation of mitochondrial MMP (mtMMP) and is associated with myocyte mechanical dysfunction. Myocytes were isolated and field stimulated at 1 and 4 Hz. Myocyte mechanics and calcium transient was studied using Ion-Optix system. Mitochondrial MMP-9 activation was evaluated using zymography. There was a 25% increase in 1 Hz and 40% increase in 4 Hz stimulation. We observed an increase in mtMMP activation with increase in electrical pacing compared to 0 Hz with a significant increase (p<0.05, n=3). Field stimulation at 4 Hz decreased cell re-lengthening. The levels of calcium transient were reduced with increase in contraction frequency. We conclude that electrical stimulation activates mtMMP-9 that is associated with myocyte mechanical dysfunction.

MMP-2/TIMP-2/TIMP-4 versus MMP-9/TIMP-3 in transition from compensatory hypertrophy and angiogenesis to decompensatory heart failure.

Although matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) play a vital role in tumour angiogenesis and TIMP-3 caused apoptosis, their role in cardiac angiogenesis is unknown. Interestingly, a disruption of co-ordinated cardiac hypertrophy and angiogenesis contributed to the transition to heart failure, however, the proteolytic and anti-angiogenic mechanisms of transition from compensatory hypertrophy to decompensatory heart failure were unclear. We hypothesized that after an aortic stenosis MMP-2 released angiogenic factors during compensatory hypertrophy and MMP-9/TIMP-3 released anti-angiogenic factors causing decompensatory heart failure. To verify this hypothesis, wild type (WT) mice were studied 3 and 8 weeks after aortic stenosis, created by banding the ascending aorta in WT and MMP-9-/- (MMP-9KO) mice. Cardiac function (echo, PV loops) was decreased at 8 weeks after stenosis. The levels of MMP-2 (western blot) increased at 3 weeks and returned to control level at 8 weeks, MMP-9 increased only at 8 weeks. TIMP-2 and -4 decreased at 3 and even more at 8 weeks. The angiogenic VEGF increased at 3 weeks and decreased at 8 weeks, the anti-angiogenic endostatin and angiostatin increased only at 8 weeks. CD-31 positive endothelial cells were more intensely labelled at 3 weeks than in sham operated or in 8 weeks banded mice. Vascularization, as estimated by x-ray angiography, was increased at 3 weeks and decreased at 8 weeks post-banding. Although the vast majority of studies were performed on control WT mice only, interestingly, MMP9-KO mice seemed to have increased vascular density 8 weeks after banding. These results suggested that there was increase in MMP-2, decrease in TIMP-2 and -4, increase in angiogenic factors and vascularization in compensatory hearts. However, in decompensatory hearts there was increase in MMP-9, TIMP-3, endostatin, angiostatin and vascular rarefaction.

Hydrogen sulfide protects against vascular remodeling from endothelial damage.

Remodeling by its very nature implied synthesis and degradation of extracellular matrix (ECM) proteins. Although oxidative stress, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) have been implicated in vascular remodeling, the differential role of MMPs versus TIMPs and oxidative stress in vascular remodeling was unclear. TIMP-3 induced vascular cell apoptosis, therefore, we hypothesized that during vascular injury TIMP-3, MMP-9 and -12 (elastin-degrading MMP) were increased, whereas MMP-2 (constitutive MMP) and TIMP-4 (cardioprotective TIMP) decreased. Because of the potent anti-oxidant, vasorelaxing, anti-hypertensive agent, hydrogen sulfide (H2S) was used to mitigate the vascular remodeling due to the differential expression of MMP and TIMP. Carotid artery injury was created by inserting a PE-10 catheter and rotating several times before pulling out. The insertion hole was sealed. Mice were grouped: wild type (WT), wild-type damaged artery (WTD), WT+NaHS (sodium hydrogen sulfide, precursor of H2S) treatment (30 µmol/L in drinking water/6 weeks) and WTD+NaHS treatment. Carotid arteries were analyzed for oxidative stress and remodeling, by measuring super oxide dismutase-1 (SOD1), p47 (NADPH oxidase subunit), nitrotyrosine, MMPs and TIMPs by in situ immunolabeling and by Western blot analyses. The results suggested robust increase in p47, nitrotyrosine, MMP-9, MMP-12, TIMP-3 and decrease in SOD1 and MMP-2 levels in the injured arteries. The treatment with H2S ameliorated these effects. We concluded that p47, TIMP-3, MMP-9 and -12 were increased where as SOD-1, MMP-2 and TIMP-4 were decreased in the injured arteries. The treatment with H2S mitigated the vascular remodeling by normalizing the levels of redox stress, MMPs and TIMPs.