CPAP failure in the management of preterm neonates with respiratory distress syndrome where surfactant is scarce. A prospective observational study.

BACKGROUND: The mainstay in the management of preterm neonates with respiratory distress syndrome (RDS) include early Continuous Positive Airway Pressure (CPAP), timely surfactant replacement and mechanical ventilation. Preterm neonates with RDS who fail CPAP are at higher risk for chronic lung disease as well as death. Unfortunately, in low resource settings CPAP may be the only treatment available for these neonates. OBJECTIVE: To determine the prevalence of CPAP failure among premature newborns with RDS and associated factors. METHODS: We conducted a prospective observational study over the first 72 h of life on 174 preterm newborns with RDS receiving CPAP at Muhimbili National Hospital (MNH). At MNH newborns with Silverman Andersen Score (SAS) of ≥ 3 are commenced on CPAP; surfactant and mechanical ventilation are very scarce. Study newborns not maintaining oxygen saturation > 90% or with SAS score ≥ 6 despite being on 50% oxygen and PEEP of 6 cmH2O and those with > 2 episodes of apnoea needing stimulation or positive pressure ventilation in 24 h were considered as CPAP failure. The prevalence of CPAP failure was determined as a percentage and factors associated were determined by logistic regression. A p-value of < 0.05 was considered significant and 95% confidence interval was used. RESULTS: Of the enrolled newborns, 48% were male and 91.4% were in-born. The mean gestational age and weight were 29 weeks (range 24-34 weeks) and 1157.7 g (range 800-1500 g) respectively. Of the mothers 44 (25%) received antenatal corticosteroids. Overall CPAP failure was 37.4% and among those weighing ≤ 1200g, it was 44.1% . Most failure occurred within the first 24 h. No factor was identified to be independently associated with CPAP failure. Mortality among those who failed CPAP was 33.8% and 12.8% among those who did not. CONCLUSIONS: In resource limited settings like ours with low up take of antenatal corticosteroids and scarce surfactant replacement a significant portion of preterm neonates especially those weighing ≤ 1200 g with RDS fail CPAP therapy.

Early outcomes of preterm neonates with respiratory distress syndrome admitted at Muhimbili National Hospital, a prospective study.

BACKGROUND: Respiratory distress syndrome (RDS) is one of the commonest complication preterm neonates suffer and accounts for a significant morbidity and mortality in low and middle income countries (LMICs). Addressing RDS is therefore crucial in reducing the under 5 mortality in LMICs. This study aimed at describing early outcomes (death/survival) of preterm neonates with RDS and identify factors associated with the outcomes among neonates admitted at Muhimbili national hospital, Tanzania. METHODS: Between October 2019 and January 2020 we conducted a prospective study on 246 preterm neonates with RDS at Muhimbili National Hospital. These were followed up for 7 days. We generated Kaplan-Meier survival curve to demonstrate time to death. We performed a cox regression analysis to ascertain factors associated with outcomes. The risk of mortality was analyzed and presented with hazard ratio. Confidence interval of 95% and P-value less than 0.05 were considered as significant. RESULTS: Of the 246 study participants 51.6% were male. The median birth weight and gestational age of participants (Inter-Quartile range) was 1.3 kg (1.0, 1.7) and 31 weeks (29, 32) respectively. Majority (60%) of study participants were inborn. Only 11.4% of mothers of study participants received steroids. Of the study participants 49 (20%) received surfactant. By day 7 of age 77/246 (31.3%) study participants had died while the majority of those alive 109/169 (64.5%) continued to need some respiratory support. Factors independently associated with mortality by day 7 included birth weight of < 1500 g (AHR = 2.11 (1.16-3.85), CI95%; p = 0.015), lack of antenatal steroids (AHR = 4.59 (1.11-18.9), CI95%; p = 0.035), 5th minute APGAR score of < 7 (AHR = 2.18 (1.33-3.56), CI95%; p = 0.002) and oxygen saturation < 90% at 6 hours post admission (AHR = 4.45 (1.68-11.7), CI95%; p = 0.003). CONCLUSION: Our study reports that there was high mortality among preterm neonates admitted with RDS mainly occurring within the first week of life. Preterm neonates with very low birth weight (VLBW), whose mother did not receive antenatal steroid, who scored < 7 at 5th minute and whose saturation was < 90% at 6 hours were at higher risk of dying. There is need to scale up antenatal corticosteroids, neonatal resuscitation training and saturation monitoring among preterm neonates with RDS.

Limited resources restrict the provision of adequate neonatal respiratory care in the countries of Africa.

AIM: Over two thirds of newborn deaths occur in Africa and South Asia, and respiratory failure is a major contributor of these deaths. The exact availability of continuous positive airway pressure (CPAP) and surfactant in Africa is unknown. The aim of this study was to describe the availability of newborn respiratory care treatments in the countries of Africa. METHODS: Surveys, in English, French and Portuguese, were sent to neonatal leaders in all 48 continental countries and the two islands with populations over 1 million. RESULTS: Forty-nine (98%) countries responded. Twenty-one countries reported less than 50 paediatricians, and 12 countries had no neonatologists. Speciality neonatal nursing was recognised in 57% of countries. Most units were able to provide supplemental oxygen. CPAP was available in 63% and 67% of the most well-equipped government and private hospitals. Surfactant was available in 33% and 39% of the most well-equipped public and private hospitals, respectively. Availability of CPAP and surfactant was greatly reduced in smaller cities. Continuous oxygen saturation monitoring was only available in 33% of countries. CONCLUSION: The availability of proven life-saving interventions in Africa is inadequate. There is a need to sustainably improve availability and use of these interventions.

Growth of preterm very low birth weight infants discharged with weight of less than 1500grams.

Early discharge of preterm very low birth weight (VLBW) infants is at times inevitable in low resource settings. The implication of such practice on the growth of this high-risk population is not known. We conducted a retrospective chart review to describe the growth of preterm VLBW infants discharged with a weight of less than 1500 g. OBJECTIVES: To describe the growth of discharged preterm VLBW infants over the first 12 weeks. METHOD: Between June 2013 and January 2014; 164 discharged preterm VLBW infants were followed up for 3 months. Among the survivors (132), we identified 111 infant records for this study. Relevant data was entered in STATA for analysis. Growth percentiles were determined at approximately 4 weeks, 8 weeks, and 12 weeks post-discharge using the intergrowth 21st growth charts. Growth velocities were computed using the 2-point average weight model. Regression analysis was used to identify factors associated with growth failure. Growth failure was defined as occipital frontal circumference (OFC), weight, and length < 10th centile by 12 weeks post-discharge. P-value of < 0.05 was considered significant at a 95% confidence interval. RESULTS: Among the study infants the median gestational age and weight at birth were 32 weeks (range 28-35 weeks) and 1250 g(range 850-1500 g) respectively; 60/111(54%) were Small for Gestational Age (SGA). The median discharge postmenstrual age (PMA) was 34 weeks (range 30-38 weeks) and weight was 1140 g (range 830-1490 g). The majority 88.2% had not recovered birth weight at discharge of whom 59.1% recovered by 2 weeks and 40.9% recovered between 2 and 4 weeks after discharge. By 12 weeks post-discharge the median PMA and weight were 46 weeks (range 37-51 weeks),and 3110 g (range 1750-5000 g) respectively, 38.7% of the infants had growth failure and 36.9% had OFC <3rd centile. Growth velocity < 15 g/kg/d in the first 4 weeks (OR 3.8, p 0.010) and subsequent 4 weeks (OR 2.5, p 0.049) post-discharge were independently associated with growth failure. CONCLUSION: Slow birth weight recovery was observed and growth failure was prevalent by 12 weeks post-discharge with more than a third having severe microcephaly. Poor post-discharge growth velocity was associated with subsequent growth failure. RECOMMENDATIONS: Growth velocity monitoring among preterm VLBW infants should be emphasized. The implication and interventions of this early growth failure needs to be explored.

Mortality among very low birth weight infants after hospital discharge in a low resource setting.

BACKGROUND: Early discharge of very low birth weight infant (VLBW) in low resource settings is inevitable but to minimize mortality of these infants after discharge we need to identify the death attributes. METHOD: A prospective cohort was conducted among 190 VLBW infants discharged from Mulago Special Care Baby Unit (SCBU) with discharge weight of < 1500 g over an 8 months period. These infants were followed up with the aims of determining the proportion dead 3 months after discharge, identifying factors associated and possible causes of death. Relevant data were captured, transferred in to STATA and imported to SPSS 12.0.1 for analysis. To determine factors associated with mortality bi-variable and multivariable regressions were conducted. A p-value of < 0.05 was considered significant and 95% confidence interval was used. RESULTS: Of the enrolled infants 164 (86.3%) completed follow up. The median gestational age of study participants was 32 weeks (range 26-35 weeks), the mean discharge weight was 1119 g (range 760-1470 g), and 59.8% were small for gestational age (SGA). During follow up 32 (19.5%) infants died. Infants discharged with weight of < 1200 g accounted for 81.2% of the deaths. Majority of the deaths (68.7%) occurred in the first month after discharge. Factors independently associated with mortality were discharge weight < 1000 g (OR 3.10, p 0.015) and not being SGA (OR 3.54, p 0.019). The main causes of death were presumed sepsis 50.0% and suspected cot death (25.0%). CONCLUSION: Mortality after hospital discharge among VLBW infants is high. Discharge at weight < 1200 g may not be a safe practice. Measures to prevent sepsis and suspected cot death should be addressed prior to considering early discharge of these infants.

Mechanisms by which calcium receptor stimulation modifies electromechanical coupling in isolated ventricular cardiomyocytes.

The calcium-sensing receptor (CaR) is widely expressed throughout the entire cardiovascular system and is capable of activating signaling pathways in different cells. Alongside calcium, the CaR also responds to physiological polycations such as putrescine underlining a participation in physiological and pathophysiological processes. Here, we aimed to determine mechanisms as to how CaR activation affects the contractile responsiveness of ventricular cardiomyocytes under basal and stimulated conditions. For that purpose, cardiac myocytes from 3-month-old male Wistar rats were isolated, and the acute effects of an antagonist (NPS2390), agonists (putrescine and gadolinium), or of downregulation of the CaR by siRNA on cell shortening were recorded in a cell-edge-detection system. In addition, experiments were performed on muscle stripes and Langendorff preparations. Mechanistic insights were taken from calcium transients of beating fura-2 AM-loaded cardiomyocytes and western blots. Isolated ventricular cardiomyocytes constitutively express CaR. The expression in the atria is less pronounced. Acute inhibition of CaR reduced basal cell shortening of ventricular myocytes at nearly physiological levels of extracellular calcium. Inhibition of CaR strongly reduced contractility of ventricular muscle stripes but not of atria. Activation of CaR by putrescine and gadolinium influences the contractile responsiveness of isolated cardiomyocytes. Increased calcium mobilization from the sarcoplasmic reticulum via an IP3-dependent mechanism was responsible for amplified systolic calcium transients and a subsequent improvement in cell shortening. Alongside with these effects, activation of CaR increased relaxation velocity of the cells. In conclusion, ventricular CaR expression affects contractile parameters of ventricular heart muscle cells and modifies electromechanical coupling of cardiomyocytes.

Mechanisms involved in postconditioning protection of cardiomyocytes against acute reperfusion injury.

Experimental and clinical studies demonstrated that postconditioning confers protection against myocardial ischemia/reperfusion injury. However the underlying cellular mechanisms responsible for the beneficial effect of postconditioning are still poorly understood. The aim of the present study was to examine the role of cytosolic and mitochondrial Ca(2+)-handling. For this purpose adult rat cardiomyocytes were subjected to simulated in vitro ischemia (glucose-free hypoxia at pH6.4) followed by simulated reperfusion with a normoxic buffer (pH7.4; 2.5 mmol/L glucose). Postconditioning, i.e., 2 repetitive cycles of normoxic (5s) and hypoxic (2.5 min) superfusion, was applied during the first 5 min of reoxygenation. Mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial Ca(2+) concentrations, cytosolic pH and necrosis were analysed applying JC-1, fura-2, fura-2/manganese, BCECF and propidium iodide, respectively. Mitochondrial permeability transition pore (MPTP) opening was detected by calcein release. Hypoxic treatment led to a reduction of ΔΨm, an increase in cytosolic and mitochondrial Ca(2+) concentration, and acidification of cardiomyocytes. During the first minutes of reoxygenation, ΔΨm transiently recovered, but irreversibly collapsed after 7 min of reoxygenation, which was accompanied by MPTP opening. Simultaneously, mitochondrial Ca(2+) increased during reperfusion and cardiomyocytes developed spontaneous cytosolic Ca(2+) oscillations and severe contracture followed by necrosis after 25 min of reoxygenation. In postconditioned cells, the collapse in ΔΨm as well as the leak of calcein, the increase in mitochondrial Ca(2+), cytosolic Ca(2+) oscillations, contracture and necrosis were significantly reduced. Furthermore postconditioning delayed cardiomyocyte pH recovery. Postconditioning by hypoxia/reoxygenation was as protective as treatment with cyclosporine A. Combining cyclosporine A and postconditioning had no additive effect. The data of the present study demonstrate that postconditioning by hypoxia/reoxygenation prevents reperfusion injury by limiting mitochondrial Ca(2+) load and thus opening of the MPTP in isolated cardiomyocytes. These effects seem to be supported by postconditioning-induced delay in pH recovery and suppression of Ca(2+) oscillations.

"I abandoned my job to look after my baby." Understanding the unpriced cost of care of a preterm infant: Caregivers' lived experiences.

BACKGROUND: Preterm birth is associated with life-long cost implications on the infant, family, health system, and society at large. The costs related to lost productivity at contributions at work during care of preterm infants are difficult to measure. We aimed to explore and document the unpriced costs parents incur following birth of a preterm infant in the first year of life in a low resource setting. METHODS: Thirty-nine mothers and five fathers of preterm infants who had ever attended the preterm follow-up clinic after discharge from Mulago National Referral Hospital, were included in a qualitative study between November 2019 and February 2020. Participants were purposively selected, and data were collected using four focused group discussions with mothers and in-depth interviews with the fathers lasting 30-70 minutes each. These were audio-recorded, transcribed and translated. The data were manually analysed using the thematic approach. FINDINGS: Three themes were generated: i) complex nature of the infant, ii) time to care for the infant, iii) mother as the predominant caregiver. The parents perceived preterm infants as delicate, complicated and their care more costly compared to those born at term. Expressions of need for time to care for their infants, frequent hospital visits and readmission were raised. Availability of the mother as the predominant caregiver some of whose roles cannot be delegated and their experiences following return to work after birth of a preterm were cited by the participants. CONCLUSION: The results highlight the unpriced costs incurred by the parents through disruption of the work pattern due to the actual and perceived needs of a preterm infant and time to care in a low resource setting. We recommend guidance on financial planning, development of policies and programs on social and financial support for parents and future studies on indirect costs of preterm care.

Late diagnosis of congenital diaphragmatic hernia: a case report.

BACKGROUND: Congenital diaphragmatic hernia beyond the neonatal period is not uncommon. Its diagnosis in infancy and early childhood poses a challenge owing to different clinical presentation ranging from gastrointestinal to respiratory symptoms. These neonates are usually misdiagnosed as having pneumonia until radiological imaging picks up the defect during routine scan for worsening respiratory symptoms. In high-income countries, the survival rate for these patients has been reported to be high, while in Sub-Saharan Africa the survival rate is still low due to delayed diagnosis, delayed referral, and hence delayed management. CASE REPORT: We present an African male baby from non-consanguineous parents, 6 weeks old, diagnosed with congenital diaphragmatic hernia at 6 weeks of age after failure to respond to antibiotics for suspected pneumonia. Despite attempts at management, he died at 5 weeks post surgery. CONCLUSION: Our case emphasizes the importance of early clinical suspicion and early detection for a differential diagnosis of congenital diaphragmatic hernia in infants who present with respiratory symptoms not responding to antibiotics or recurrent pneumonia, and improving the availability of imaging in primary care facilities to diagnose such defects early and manage them accordingly.

Esophageal atresia with tracheoesophageal fistula: two case reports.

BACKGROUND: The incidence of esophageal atresia with tracheoesophageal fistula is 1 out of 3000-5000 live births. Its incidence in lower middle income countries is not known. The infants usually present with excessive secretions or choking while feeding and are at risk for aspiration. The outcome of these infants in lower middle income countries is not encouraging due to delays in referral, sepsis at presentation requiring preoperative stabilization, postoperative complications such as anastomosis leaks, pneumonia, and pneumothorax. CASE PRESENTATION: We present two African babies who were term infants at age 2 days (male) and 5 days (female) with diagnosis of esophageal atresia and tracheoesophageal fistula. The 5-day-old infant required preoperative stabilization due to sepsis and delayed surgery with a poor postoperative outcome. The 2-day-old infant was preoperatively stable and had a good postoperative outcome. The challenges faced in management of these two cases have been highlighted. CONCLUSION: Outcome of infants with esophageal atresia and tracheoesophageal fistula in lower middle income countries is not encouraging due to delays in referral and poor postoperative healing attributed to sepsis and recurrent pneumothorax. Timely referral, preoperative condition of the infant, and timely management has shown to be a contributory factor for an improved outcome.

Opposing effects of ATP and adenosine on barrier function of rat coronary microvasculature.

ATP can differentially affect the micro- and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular endothelial cells and microvessels, in vivo. We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular endothelial monolayers (RCEC) in vitro, as well as in rat mesentery vessels, and in rat hearts in vivo. ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracellular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5'-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A(1), and A(2) but not by A(3) receptor antagonists. Likewise, immunofluorescence microscopy of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo. In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes.

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

TGFß receptor activation enhances cardiac apoptosis via SMAD activation and concomitant NO release.

UNLABELLED: Transforming growth factor ß (TGFß) expression is induced in the myocardium during transition from compensated hypertrophy to heart failure. In cardiomyocytes, stimulation with TGFß results in restricted contractile function and enhanced apoptosis. Nitric oxide (NO) also induces apoptosis and influences cardiac function. Therefore, we wanted to know whether NO is causally involved in TGFß-induced apoptosis. In isolated ventricular cardiomyocytes of adult rat incubation with TGFß(1) increased NO release which was inhibited by NOS inhibitor ETU but not with iNOS inhibitor (1400 W) or nNOS inhibitor (TFA). In addition, TGFß-induced apoptosis was blocked with ETU and ODQ, but not with 1400 W or TFA. The consequent assumption that endothelial NOS is involved in TGFß-induced NO formation and apoptosis was supported by increased phosphorylation of eNOS at serine 1177 and by the fact that TGFß did not increase NO release in eNOS KO mice. Furthermore, TGFß-induced apoptosis, NO formation, SMAD binding activity and SMAD2 phosphorylation were blocked by a TGFß receptor antagonist, but only apoptosis and NO formation could be blocked with ETU. Expression of SMAD7 was increased after TGFß stimulation and blocked with TGFß receptor antagonist but not after blocking NO synthase with ETU. CONCLUSION: In cardiomyocytes TGFß-induced apoptosis is mediated via TGFß receptor activation that concomitantly activates SMAD transcription factors and the eNOS/NO/sGC pathway. Both of these pathways are needed for apoptosis induction by TGFß. This reveals a new pathway of cardiac NO release and identifies NO as a possible contributor to heart failure progression mediated by TGFß.

Aldosterone improves contractile function of adult rat ventricular cardiomyocytes in a non-acute way: potential relationship to the calcium paradox of aldosteronism.

Heart failure is accompanied by electrolyte disturbance including reduced calcium and sodium in the extracellular milieu but increased calcium within cells, a phenomenon called "calcium paradox". Aldosteronism is considered as part of this disorder. Aldosterone antagonism is known to reduce cardiac mortality on top of standard therapies such as antagonism of the renin-angiotensin-system. However, the effect of aldosterone on cardiac function under basal conditions and conditions more closely related to those seen in heart failure remains elusive. In order to address this question the function of isolated cardiomyocytes was determined as unloaded cell shortening. Cardiomyocytes were isolated from adult rat hearts and cultured for 24 h in the presence of aldosterone. Thereafter, cell shortening was determined in cells that were electrically paced (0.5-2.0 Hz). The effect of aldosterone on cell shortening was investigated under basal and maximal inotropic stimulation, preincubation with angiotensin II and myocytes from spontaneously hypertensive rats. The composition of the culture medium was modified according to the extracellular milieu found in patients with end-stage heart failure. Aldosterone increased cell shortening in a frequency-dependent way under basal conditions and conditions of low calcium. It potentiated the effect of beta-adrenoceptor stimulation, increased the formation of oxygen radicals, and increased diastolic and systolic calcium. In conclusion, chronic exposure to aldosterone improves the function of cardiomyocytes under basal conditions and electrolyte disturbances that mimic the situation found in heart failure patients.

Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration.

Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. The Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10-100 µmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10-100 µM) on the membrane potential was measured with the fluorescence dye DiBAC4(3) and the effect on [Ca+2]i with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10-100 µmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 µmol/L (35.1±1.8% as compared to control (p≤0.05; n=45)). As compared to the control, the addition of BbCI (100 µmol/L) caused a significant increase of systolic Ca2+ of 28.4±5.0% after 30 min incubation. HUVEC treatment with BbCI (100 µmol/L) showed a weak but significant decrease of the membrane potential of 9.5±0.9% as compared to control (p≤0.05; n=80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca2+ concentration and the membrane potential.

Protective role of soluble adenylyl cyclase against reperfusion-induced injury of cardiac cells.

AIMS: Disturbance of mitochondrial function significantly contributes to the myocardial injury that occurs during reperfusion. Increasing evidence suggests a role of intra-mitochondrial cyclic AMP (cAMP) signaling in promoting respiration and ATP synthesis. Mitochondrial levels of cAMP are controlled by type 10 soluble adenylyl cyclase (sAC) and phosphodiesterase 2 (PDE2), however their role in the reperfusion-induced injury remains unknown. Here we aimed to examine whether sAC may support cardiomyocyte survival during reperfusion. METHODS AND RESULTS: Adult rat cardiomyocytes or rat cardiac H9C2 cells were subjected to metabolic inhibition and recovery as a model of simulated ischemia and reperfusion. Cytosolic Ca2+, pH, mitochondrial cAMP (live-cell imaging), and cell viability were analyzed during a 15-min period of reperfusion. Suppression of sAC activity in cardiomyocytes and H9C2 cells, either by sAC knockdown, by pharmacological inhibition or by withdrawal of bicarbonate, a natural sAC activator, compromised cell viability and recovery of cytosolic Ca2+ homeostasis during reperfusion. Contrariwise, overexpression of mitochondria-targeted sAC in H9C2 cells suppressed reperfusion-induced cell death. Analyzing cAMP concentration in mitochondrial matrix we found that inhibition of PDE2, a predominant mitochondria-localized PDE isoform in mammals, during reperfusion significantly increased cAMP level in mitochondrial matrix, but not in cytosol. Accordingly, PDE2 inhibition attenuated reperfusion-induced cardiomyocyte death and improved recovery of the cytosolic Ca2+ homeostasis. CONCLUSION: sAC plays an essential role in supporting cardiomyocytes viability during reperfusion. Elevation of mitochondrial cAMP pool either by sAC overexpression or by PDE2 inhibition beneficially affects cardiomyocyte survival during reperfusion.

The role of poly(ADP-ribose) polymerase (PARP) in the autonomous proliferative response of endothelial cells to hypoxia.

OBJECTIVE: The autonomous proliferative response of endothelial cells to hypoxia has been shown to be dependent on activation of NAD(P)H oxidase, on the cytosolic Ca2+ load, and, consequently, on nuclear translocation of extracellular signal-regulated kinase (ERK)1/2 during transient hypoxia. The aim of the present study was to investigate whether poly(ADP-ribose) polymerase (PARP) is a downstream signal of NAD(P)H oxidase, mediating cytosolic Ca2+ load and hence nuclear translocation of ERK1/2 and endothelial cell proliferation. METHODS: Porcine aortic endothelial cells were incubated under hypoxic conditions for 40 min. Cytosolic [Ca2+] and reactive oxygen species (ROS) formation were measured in fura-2- and DCF-loaded cells, respectively. PARP activation was detected by immunocytochemistry, and endothelial cell proliferation was determined 24 h after 60 min of transient hypoxia. RESULTS: Inhibition of NAD(P)H oxidase with antisense oligonucleotide against the p22(phox) subunit, MEK/ERK signalling with UO 126 (30 microM), or PARP with PJ 34 (10 microM) leads to a marked reduction in hypoxia-induced cytosolic Ca2+ load and activation of PARP. Hypoxia-induced translocation of ERK1/2 and endothelial cell proliferation were also prevented when NAD(P)H oxidase or PARP were inhibited; however, hypoxic ROS formation was not affected in the presence of PARP inhibitor. CONCLUSION: PARP represents a downstream effector of NADP(H) oxidase and acts as a necessary intermediate step for the hypoxic proliferative response of endothelial cells.

Ischemic acidosis causes apoptosis in coronary endothelial cells through activation of caspase-12.

OBJECTIVE: Myocardial ischemia has been shown to induce apoptosis of endothelial cells (EC). However, the mechanism of this endothelial injury is still poorly understood. To analyse the signaling pathway of ischemia-induced EC apoptosis was the aim of the present study. METHODS: The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pH(o) 6.4). Apoptosis was defined by staining of nuclei with Hoechst-33342 and TUNEL. Cytosolic Ca2+ and pH were measured with Fura-2 and BCECF, respectively. RESULTS: Apoptosis (29.2+/-1.7% of cells) induced by exposure to simulated ischemia for 2 h was accompanied by cytosolic Ca2+ overload (1090+/-52 nmol/l) and acidosis (pHi = 6.52+/-0.13). Simulated ischemia had no significant effect on caspase-8 cleavage, but induced cleavage of caspase-3 and caspase-12 and led to a slight release of cytochrome C. Prevention of cytosolic acidosis (anoxia at pH(o) 7.4) had no effect on cytochrome C release, but significantly reduced apoptosis, attenuated cytosolic Ca2+ overload, and prevented cleavage of caspase-12. A similar effect was achieved by inhibition of Ca2+ release channels in the endoplasmic reticulum with ryanodine and xestospongin C. Knock-down of caspase-12 with small interfering RNA suppressed caspase-3 activation and reduced apoptotic cell number by about 70%. CONCLUSION: Acidosis, rather than anoxia, is an important trigger of apoptosis in EC under simulated ischemia. The main pathway of the simulated ischemia-induced apoptosis consists of the Ca2+ leak from the ER followed by activation of caspase-12 and caspase-3.

Activation of Ca2+ -activated potassium channels is involved in lysophosphatidylcholine-induced monocyte adhesion to endothelial cells.

OBJECTIVE: Ca(2+)-activated K(+)-channels (BK(Ca)) play an important role in lysophosphatidylcholine (LPC)-induced endothelial dysfunction. Aim of our study was to investigate whether LPC-induced activation of BK(Ca) is also involved in monocyte adhesion to endothelial cells (EC). METHODS AND RESULTS: Measurement of membrane potential (MP) was performed using the fluorescence dye DiBAC. Adhesion of the monocytotic cell line U937 to EC was analysed by (3)[H]-thymidine-adhesion-assay. Expression of ICAM-1 and VCAM-1 were analyzed by FACS. LPC induced a hyperpolarization of EC in a dose-dependent manner with the maximum seen with 2 microM. This was prevented by the BK(Ca)-inhibitor iberiotoxin (IBX, 100nM). Adhesion of U937 cells to EC was increased after stimulation of EC with LPC. This effect was time-dependent with the maximum seen after 4h. LPC-induced adhesion was significantly reduced when EC were co-incubated with IBX, or NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI, 5 microM) and also blocked by addition of 2-aminoethoxydiphenylborate (2-APB, 100 microM) or the calcium-chelator BAPTA (10 microM). Stimulation of U937 cells with LPC did not result in an increased adhesion to unstimulated EC. CONCLUSION: Activation of the endothelial BK(Ca) plays an important role in monocyte adhesion to endothelial cells.

Contribution of PI 3-kinase isoforms to angiotensin II- and alpha-adrenoceptor-mediated signalling pathways in cardiomyocytes.

OBJECTIVE: Angiotensin II stimulation increases the formation of reactive oxygen species (ROS), the phosphorylation of p38 mitogen-activated protein kinase (MAPK), and the expression of transforming growth factor beta (TGFbeta) in adult cardiomyocytes. The aim of this study was to determine the involvement of PI 3-kinase and to specify the participation of different isoforms in the angiotensin II-induced formation of ROS in comparison to the hypertrophic pathway triggered by alpha-adrenoceptor stimulation. METHODS: Freshly isolated myocytes were used to examine formation of ROS via H(2)DCF fluorescence. p38 MAPK phosphorylation, p70(S6)-kinase phosphorylation, PI 3-kinase, and TGFbeta expression were measured by Western blotting. Sense and antisense oligonucleotides were used to down-regulate diverse PI 3-kinase isoforms. Hypertrophy was measured by (14)C-phenylalanine incorporation and cell volume. RESULTS: Inhibition of PI 3-kinase by Ly294002 or wortmannin, two inhibitors, decreased formation of ROS, phosphorylation of p38 MAPK, and TGFbeta expression. Down-regulation of the p110beta isoform by antisense oligonucleotides inhibited the angiotensin II-induced signalling pathway but not the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes. In contrast, down-regulation of the p110alpha isoform decreased the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes but did not affect the angiotensin II-mediated signalling pathway. CONCLUSION: Thus, our study identifies an involvement of PI 3-kinase in the angiotensin II-induced formation of ROS and provides a biochemical basis for ligand-specific responses for angiotensin II and alpha-adrenoceptor stimulation as relates to hypertrophy.