Sex-specific responses to slow progressive pressure overload in a large animal model of HFpEF.

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.

Regenerative therapy based on miRNA-302 mimics for enhancing host recovery from pneumonia caused by Streptococcus pneumoniae.

Bacterial pneumonia remains a leading cause of morbidity and mortality worldwide. A defining feature of pneumonia is lung injury, leading to protracted suffering and vulnerability long after bacterial clearance. Little is known about which cells are damaged during bacterial pneumonia and if the regenerative process can be harnessed to promote tissue repair and host recovery. Here, we show that infection of mice with Streptococcus pneumoniae (Sp) caused substantial damage to alveolar epithelial cells (AEC), followed by a slow process of regeneration. Concurrent with AEC regeneration, the expression of miRNA-302 is elevated in AEC. Treatment of Sp-infected mice with miRNA-302 mimics improved lung functions, host recovery, and survival. miRNA-302 mediated its therapeutic effects, not by inhibiting apoptosis and preventing damage, but by promoting proliferation of local epithelial progenitor cells to regenerate AEC. These results demonstrate the ability of microRNA-based therapy to promote AEC regeneration and enhance host recovery from bacterial pneumonia.

Protein kinase C-delta inhibition is organ-protective, enhances pathogen clearance, and improves survival in sepsis.

Sepsis is a leading cause of morbidity and mortality in intensive care units. Previously, we identified Protein Kinase C-delta (PKCδ) as an important regulator of the inflammatory response in sepsis. An important issue in development of anti-inflammatory therapeutics is the risk of immunosuppression and inability to effectively clear pathogens. In this study, we investigated whether PKCδ inhibition prevented organ dysfunction and improved survival without compromising pathogen clearance. Sprague Dawley rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Post-surgery, PBS or a PKCδ inhibitor (200µg/kg) was administered intra-tracheally (IT). At 24 hours post-CLP, there was evidence of lung and kidney dysfunction. PKCδ inhibition decreased leukocyte influx in these organs, decreased endothelial permeability, improved gas exchange, and reduced blood urea nitrogen/creatinine ratios indicating organ protection. PKCδ inhibition significantly decreased bacterial levels in the peritoneal cavity, spleen and blood but did not exhibit direct bactericidal properties. Peritoneal chemokine levels, neutrophil numbers, or macrophage phenotypes were not altered by PKCδ inhibition. Peritoneal macrophages isolated from PKCδ inhibitor-treated septic rats demonstrated increased bacterial phagocytosis. Importantly, PKCδ inhibition increased survival. Thus, PKCδ inhibition improved survival and improved survival was associated with increased phagocytic activity, enhanced pathogen clearance, and decreased organ injury.

Nebulization of Single-Chain Tissue-Type and Single-Chain Urokinase Plasminogen Activator for Treatment of Inhalational Smoke-Induced Acute Lung Injury.

Single-chain tissue-type plasminogen activator (sctPA) and single-chain urokinase plasminogen activator (scuPA) have attracted interest as enzymes for the treatment of inhalational smoke-induced acute lung injury (ISALI). In this study, the pulmonary delivery of commercial human sctPA and lyophilized scuPA and their reconstituted solution forms were demonstrated using vibrating mesh nebulizers (Aeroneb® Pro (active) and EZ Breathe® (passive)). Both the Aeroneb® Pro and EZ Breathe® vibrating mesh nebulizers produced atomized droplets of protein solution of similar size of less than about 5 µm, which is appropriate for pulmonary delivery. Enzymatic activities of scuPA and of sctPA were determined after nebulization and both remained stable (88.0% and 93.9%). Additionally, the enzymatic activities of sctPA and tcuPA were not significantly affected by excipients, lyophilization or reconstitution conditions. The results of these studies support further development of inhaled formulations of fibrinolysins for delivery to the lungs following smoke-induced acute pulmonary injury.

Biodistribution and Efficacy of Targeted Pulmonary Delivery of a Protein Kinase C-δ Inhibitory Peptide: Impact on Indirect Lung Injury.

Sepsis and sepsis-induced lung injury remain a leading cause of death in intensive care units. We identified protein kinase C-δ (PKCδ) as a critical regulator of the acute inflammatory response and demonstrated that PKCδ inhibition was lung-protective in a rodent sepsis model, suggesting that targeting PKCδ is a potential strategy for preserving pulmonary function in the setting of indirect lung injury. In this study, whole-body organ biodistribution and pulmonary cellular distribution of a transactivator of transcription (TAT)-conjugated PKCδ inhibitory peptide (PKCδ-TAT) was determined following intratracheal (IT) delivery in control and septic [cecal ligation and puncture (CLP)] rats to ascertain the impact of disease pathology on biodistribution and efficacy. There was negligible lung uptake of radiolabeled peptide upon intravenous delivery [<1% initial dose (ID)], whereas IT administration resulted in lung retention of >65% ID with minimal uptake in liver or kidney (<2% ID). IT delivery of a fluorescent-tagged (tetramethylrhodamine-PKCδ-TAT) peptide demonstrated uniform spatial distribution and cellular uptake throughout the peripheral lung. IT delivery of PKCδ-TAT at the time of CLP surgery significantly reduced PKCδ activation (tyrosine phosphorylation, nuclear translocation and cleavage) and acute lung inflammation, resulting in improved lung function and gas exchange. Importantly, peptide efficacy was similar when delivered at 4 hours post-CLP, demonstrating therapeutic relevance. Conversely, spatial lung distribution and efficacy were significantly impaired at 8 hours post-CLP, which corresponded to marked histopathological progression of lung injury. These studies establish a functional connection between peptide spatial distribution, inflammatory histopathology in the lung, and efficacy of this anti-inflammatory peptide.

Pulmonary endothelial protein kinase C-delta (PKCδ) regulates neutrophil migration in acute lung inflammation.

Excessive neutrophil migration across the pulmonary endothelium into the lung and release of oxidants and proteases are key elements in pathogenesis of acute lung injury. Previously, we identified protein kinase C-delta (PKCδ) as an important regulator of proinflammatory signaling in human neutrophils and demonstrated that intratracheal instillation of a TAT-conjugated PKCδ inhibitory peptide (PKCδ-TAT) is lung protective in a rat model of sepsis-induced indirect pulmonary injury (cecal ligation and puncture). In the present study, intratracheal instillation of this PKCδ inhibitor resulted in peptide distribution throughout the lung parenchyma and pulmonary endothelium and decreased neutrophil influx, with concomitant attenuation of sepsis-induced endothelial ICAM-1 and VCAM-1 expression in this model. To further delineate the role of PKCδ in regulating neutrophil migration, we used an in vitro transmigration model with human pulmonary microvascular endothelial cells (PMVECs). Consistent with in vivo findings, inhibition of PMVEC PKCδ decreased IL-1ß-mediated neutrophil transmigration. PKCδ regulation was stimulus-dependent; PKCδ was required for transmigration mediated by IL-1ß and fMLP (integrin-dependent), but not IL-8 (integrin-independent). PKCδ was essential for IL-1ß-mediated neutrophil adherence and NF-κB-dependent expression of ICAM-1 and VCAM-1. In PMVECs, IL-1ß-mediated production of ROS and activation of redox-sensitive NF-κB were PKCδ dependent, suggesting an upstream signaling role. Thus, PKCδ has an important role in regulating neutrophil-endothelial cell interactions and recruitment to the inflamed lung.

The orl rat is more responsive to methacholine challenge than wild type.

BACKGROUND: This study presents an animal model of native airway hyperresponsiveness (AHR). AHR is a fundamental aspect of asthma and reflects an abnormal response characterized by airway narrowing following exposure to a wide variety of non-immunological stimuli. Undescended testis (UDT) is one of the most common male congenital anomalies. The orl rat is a Long Evans substrain with inherited UDT. Since boys born with congenital UDT are more likely to manifest asthma symptoms, the main aim of this study was to investigate the alternative hypothesis that orl rats have greater AHR to a methacholine aerosol challenge than wild type rats. METHODS: Long Evans wild type (n = 9) and orl (n = 13) rats were anesthetized, tracheostomized, and mechanically ventilated at 4 weeks of age. Escalating concentrations of inhaled methacholine were delivered. The methacholine potency and efficacy in the strains were measured. Respiratory resistance was the primary endpoint. After the final methacholine aerosol challenge, the short-acting ß2-adrenoceptor agonist albuterol was administered as an aerosol and lung/diaphragm tissues were assayed for interleukin (IL)-4, IL-6, and tumor necrosis factor (TNF)-α. Histological and histomorphometrical analyses were performed. RESULTS: The methacholine concentration-response curve in the orl group indicated increased sensitivity, hyperreactivity, and exaggerated maximal response in comparison with the wild type group, indicating that orl rats had abnormally greater AHR responses to methacholine. Histological findings in orl rats showed the presence of eosinophils, unlike wild type rats. ß2-Adrenoceptor agonist intervention resulted in up-regulation of IL-4 diaphragmatic levels and down-regulation of IL-4 and IL-6 in the lungs of orl rats. CONCLUSION: orl rats had greater AHR than wild type rats during methacholine challenge, with higher IL-4 levels in diaphragmatic tissue homogenates. Positive immunostaining for IL-4 was detected in lung and diaphragmatic tissue in both strains. This model offers advantages over other pre-clinical murine models for studying potential mechanistic links between cryptorchidism and asthma. This animal model may be useful for further testing of compounds/therapeutics options for treating AHR.

Lucinactant attenuates pulmonary inflammatory response, preserves lung structure, and improves physiologic outcomes in a preterm lamb model of RDS.

BACKGROUND: Acute inflammatory responses to supplemental oxygen and mechanical ventilation have been implicated in the pathophysiological sequelae of respiratory distress syndrome (RDS). Although surfactant replacement therapy (SRT) has contributed to lung stability, the effect on lung inflammation is inconclusive. Lucinactant contains sinapultide (KL4), a novel synthetic peptide that functionally mimics surfactant protein B, a protein with anti-inflammatory properties. We tested the hypothesis that lucinactant may modulate lung inflammatory response to mechanical ventilation in the management of RDS and may confer greater protection than animal-derived surfactants. METHODS: Preterm lambs (126.8 ± 0.2 SD d gestation) were randomized to receive lucinactant, poractant alfa, beractant, or no surfactant and studied for 4 h. Gas exchange and pulmonary function were assessed serially. Lung inflammation biomarkers and lung histology were assessed at termination. RESULTS: SRT improved lung compliance relative to no SRT without significant difference between SRT groups. Lucinactant attenuated lung and systemic inflammatory response, supported oxygenation at lower ventilatory requirements, and preserved lung structural integrity to a greater degree than either no SRT or SRT with poractant alfa or beractant. CONCLUSION: These data suggest that early intervention with lucinactant may more effectively mitigate pulmonary pathophysiological sequelae of RDS than the animal-derived surfactants poractant alfa or beractant.

Pregnancy and outcome of uterine allotransplantation and assisted reproduction in sheep.

This pilot study was performed from March 2008 through February 2010 to demonstrate that pregnancy can be achieved in a uterine allograft in the sheep model with the guidance of assisted reproductive technology. Uterine allotransplantation was performed in 12 sexually mature African sheep (Sudanese and Ethiopian). All animals underwent uterine transplantation via a minilaparotomy incision using a Mobius retractor device. A control group of pregnant Romney Marsh sheep with nontransplanted uteri were used to compare fetal development, uterine and placental histologic findings, and blood samples of progeny of the uterine transplant recipient sheep. Fetal size was obtained from ultrasound measurements during the early (crown-rump length) and late (biparietal diameter and abdominal circumference) gestational periods. The primary end point variables included preoperative and postoperative management, embryo transfer protocol, intraoperative assessments, and physiologic cardiopulmonary changes in the lamb during the first 5 hours of life. Four months after the initial uterine transplantation, 5 of 12 uterine allografts were considered candidates for the embryo transfer procedure. Fresh and frozen blastocyst donors were transferred accordingly to the remaining 5 uterine allografts via a minilaparotomy incision. Three of these resulted in pregnancies. One was an ectopic gestation, 1 sheep carried the pregnancy to 105 days, and 1 delivered a fully developed lamb from the transplanted uterus that was delivered via cesarean section. Neonatal lamb blood gas values and chemistry, gross organ examination, and ventilation and respiratory compliance studies yielded results normal for gestational age. This first reported case demonstrates that pregnancy can be carried in an allotransplanted uterus, with the end result a successful delivery.

Prototype hybrid systems for neonatal warming: in vitro comparisons to standard of care devices.

Preterm infants lack necessary thermoregulation. An ideal incubator should maintain a uniform and constant thermal environment. We compared the effectiveness of a supplemental heating blanket to improve the heating characteristics of two different incubator warming devices using assessment of their respective function alone as controls. Device A and device B, with and without a heating blanket (Harvard Apparatus), were instrumented with a distribution matrix of multiple temperature (n = 11) and humidity probes. These data were serially measured during warm up to 37.5 °C and through a series of open-door perturbations. The time constant, temperature variation, and change in air temperature were calculated. Data were analyzed for significance by 2-factor ANOVA for each respective incubator either turned on or off with either the heating blanket turned on or off. Device A warms faster (33.87% ; p < 0.05) than device B, but has a greater (37.27% ; p < 0.05) temperature variation during warmup. The heating blanket enhances the thermal response of device A during warmup, but does not alter those of device B. With the side door open, device A shows a smaller (-16.5% ; p < 0.05) temperature variation than device B; the heating blanket attenuates the temperature change in both devices. These results demonstrate that the use of a supplemental heating blanket, as well as device-related differences, may impact clinical control of a thermal environment.