Daniel O'Toole

Mesenchymal stem cells enhance recovery and repair following ventilator-induced lung injury in the rat

Background Bone-marrow derived mesenchymal stem cells (MSCs) reduce the severity of evolving acute lung injury (ALI), but their ability to repair the injured lung is not clear. A study was undertaken to determine the potential for MSCs to enhance repair after ventilator-induced lung injury (VILI) and elucidate the mechanisms underlying these effects. Methods Anaesthetised rats underwent injurious ventilation which produced severe ALI. Following recovery, they were given an intravenous injection of MSCs (2×106 cells) or vehicle immediately and a second dose 24 h later. The extent of recovery following VILI was assessed after 48 h. Subsequent experiments examined the potential for non-stem cells and for the MSC secretome to enhance VILI repair. The contribution of specific MSC-secreted mediators was then examined in a wound healing model. Results MSC therapy enhanced repair following VILI. MSCs enhanced restoration of systemic oxygenation and lung compliance, reduced total lung water, decreased lung inflammation and histological lung injury and restored lung structure. They attenuated alveolar tumour necrosis factor α concentrations while increasing concentrations of interleukin 10. These effects were not seen with non-stem cells (ie, rat fibroblasts). MSC-secreted products also enhanced lung repair and attenuated the inflammatory response following VILI. The beneficial effect of the MSC secretome on repair of pulmonary epithelial wounds was attenuated by prior depletion of keratinocyte growth factor. Conclusion MSC therapy enhances lung repair following VILI via a paracrine mechanism that may be keratinocyte growth factor-dependent.

Hypercapnic acidosis attenuates pulmonary epithelial wound repair by an NF-κB dependent mechanism

Background: Hypercapnic acidosis exerts protective effects in acute lung injury but may also slow cellular repair. These effects may be mediated via inhibition of nuclear factor-κB (NF-κB), a pivotal transcriptional regulator in inflammation and repair. Objectives: To determine the effects of hypercapnic acidosis in pulmonary epithelial wound repair, to elucidate the role of NF-κB and to examine the mechanisms by which these effects are mediated. Methods: Confluent small airway epithelial cell, human bronchial epithelial cell and type II alveolar A549 cell monolayers were subjected to wound injury under conditions of hypercapnic acidosis (pH 7.0, carbon dioxide tension (Pco2) 11 kPa) or normocapnia (pH 7.37, Pco2 5.5 kPa) and the rate of healing determined. Subsequent experiments investigated the role of hypercapnia versus acidosis and elucidated the role of NF-κB and mitogen-activated protein kinases. The roles of cellular mitosis versus migration and of matrix metalloproteinases in mediating these effects were then determined. Results: Hypercapnic acidosis reduced wound closure (mean (SD) 33 (6.3)% vs 64 (5.9)%, p<0.01) and reduced activation of NF-κB compared with normocapnia. Buffering of the acidosis did not alter this inhibitory effect. Prior inhibition of NF-κB activation occluded the effect of hypercapnic acidosis. Inhibition of ERK, JNK and P38 did not modulate wound healing. Hypercapnic acidosis reduced epithelial cell migration but did not alter mitosis, and reduced matrix metalloproteinase-1 while increasing concentrations of tissue inhibitor of metalloproteinase-2. Conclusions: Hypercapnic acidosis inhibits pulmonary epithelial wound healing by reducing cell migration via an NF-κB dependent mechanism that may involve alterations in matrix metalloproteinase activity.

Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

IntroductionA combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.MethodsTransduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.ResultsThe second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.ConclusionsCollectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.

Hypercapnic acidosis attenuates shock and lung injury in early and prolonged systemic sepsis

Objective:To investigate whether acute hypercapnic acidosis—induced by adding CO2 to inspired gas—would protect against severe systemic sepsis-induced lung and systemic organ injury resulting from cecal ligation and puncture. Acute hypercapnic acidosis protects against lung injury after both nonseptic and early pneumonia-induced lung injury. In contrast, prolonged hypercapnia worsens pneumonia-induced lung injury. The effects of hypercapnia and acidosis in the setting of systemic sepsis remain to be determined. Design:Prospective randomized animal study. Setting:University research laboratory. Subjects:Adult male Sprague-Dawley rats. Interventions:In the early systemic sepsis series, post induction of anesthesia and tracheostomy placement, animals were randomized to normocapnia (Fico2 = 0.00, n = 12) or hypercapnic acidosis (Fico2 = 0.05, n = 12). Cecal ligation and puncture were performed and the animals were ventilated for 3 hrs. In the prolonged systemic sepsis series, rats were anesthetized, cecal ligation and puncture were performed, and the animals were allowed to recover. The animals were then randomized to housing under conditions of environmental normocapnia (Fico2 = 0.00, n = 20) or hypercapnia (Fico2 = 0.08, n = 20). After 96 hrs, the animals were reanesthetized, and the severity of lung and hemodynamic injury was assessed. Results:In early systemic sepsis, hypercapnic acidosis attenuated the development and severity of hypotension, and reduced lactate accumulation and the decrement in central venous oxyhemoglobin levels, compared with normocapnia. Hypercapnic acidosis reduced bronchoalveolar lavage neutrophil infiltration, and lung wet/dry weight ratios. In prolonged systemic sepsis, hypercapnic acidosis reduced histologic indices of lung injury. There was no evidence that hypercapnia worsened prolonged systemic sepsis-induced lung injury. Hypercapnic acidosis did not alter lung or systemic bacterial loads in early or prolonged systemic sepsis. Conclusion:Hypercapnic acidosis exerts beneficial effects in early and prolonged cecal ligation and puncture-induced polymicrobial systemic sepsis.

Human mesenchymal stromal cells decrease the severity of acute lung injury induced by E. coli in the rat

Background Mesenchymal stromal cells (MSCs) demonstrate considerable promise in preclinical acute respiratory distress syndrome models. We wished to determine the efficacy and mechanisms of action of human MSCs (hMSCs) in the setting of acute lung injury induced by prolonged Escherichia coli pneumonia in the rat. Methods Adult male Sprague Dawley rats underwent intratracheal instillation of E. coli bacteria in all experiments. In Series 1, animals were randomised to intravenous administration of: (1) vehicle (phosphate buffered saline (PBS), 300 μL); (2) 1×107 fibroblasts/kg; (3) 1×107 hMSCs/kg or (4) 2×107 hMSCs/kg. Series 2 determined the lowest effective hMSC dose. Series 3 compared the efficacy of intratracheal versus intravenous hMSC administration, while Series 4 examined the efficacy of cryopreserved hMSC. Series 5 examined the efficacy of the hMSC secretome. Parallel in vitro experiments further assessed the potential for hMSCs to secrete LL-37 and modulate macrophage phagocytosis. Results hMSC therapy reduced the severity of rodent E. coli pneumonia, improving survival, decreasing lung injury, reducing lung bacterial load and suppressing inflammation. Doses as low as 5×106 hMSCs/kg were effective. Intratracheal hMSC therapy was as effective as intravenous hMSC. Cryopreserved hMSCs were also effective, while the hMSC secretome was less effective in this model. hMSC therapy enhanced macrophage phagocytic capacity and increased lung and systemic concentrations of the antimicrobial peptide LL37. Conclusions hMSC therapy decreased E. coli induced pneumonia injury and reduced lung bacterial burden, potentially via enhanced macrophage phagocytosis and increased alveolar LL-37 concentrations.

Infection-induced lung injury is worsened after renal buffering of hypercapnic acidosis

Objective:Prolonged hypercapnia is commonly encountered during the treatment of acute respiratory distress syndrome and acute respiratory failure attributable to other causes with protective ventilation strategies. In these circumstances, compensatory renal buffering returns pH to normal establishing a condition of buffered hypercapnia. It is also common intensive care practice to correct the pH more rapidly using bicarbonate infusions. Although it is well-established that hypercapnic acidosis has potent anti-inflammatory and protective effects, the effect of buffered hypercapnia on acute lung injury and acute respiratory distress syndrome is unknown. We therefore wished to determine the effects of buffered hypercapnia on acute lung injury induced by endotoxin or Escherichia coli infection in vivo. Design:Prospective, randomized animal study. Setting:University research laboratory. Subjects:Adult male Sprague-Dawley rats. Interventions:We established buffered hypercapnia by exposing rats to a hypercapnic environment for 3 days before the induction of lung injury. Buffered hypercapnia rats (initial pH >7.35, FiCO2 = 0.05) and normocapnic controls (initial pH >7.35, FiCO2 = 0.00) were then anesthetized, mechanically ventilated, and lung injury induced by intra-tracheal inoculation of endotoxin (series I) or Escherichia coli (series II). Measurements and Main Results:Buffered hypercapnia significantly increased both endotoxin and Escherichia coli-induced lung injury when compared to normocapnic controls, as assessed by arterial oxygenation, lung compliance, pro-inflammatory pulmonary cytokine concentrations, and measurements of structural lung damage. In additional in vitro experiments buffered hypercapnia did not alter neutrophil phagocytosis ability but did impaired epithelial wound healing. Conclusions:Our results demonstrate that infection-induced injury in vivo is worsened after renal buffering of hypercapnic acidosis independently of any changes in tidal volume. These findings have important implications for our understanding of the pathogenesis of infection-induced lung injury during the use protective ventilation strategies that permits buffered hypercapnia and during infective exacerbations of chronic lung diseases associated with sustained hypercapnia.

Mesenchymal Stem Cell Survival in the Infarcted Heart Is Enhanced by Lentivirus Vector-Mediated Heat Shock Protein 27 Expression

Mesenchymal stem cell (MSC) therapy offers the potential to promote recovery after myocardial infarction (MI). However, therapeutic efficacy may be limited by poor survival and retention of transplanted cells. A combination of gene and cell therapy has the capacity to prevent donor cell death and augment the reparative and regenerative effects of cell transfer. The present study investigates the effect of exogenous heat shock protein 27 (Hsp27) expression in MSCs in an in vitro model of ischemia and in an in vivo rat MI model and aims to determine if this could enhance the therapeutic benefit associated with cell delivery. Hsp27 overexpression by lentivirus vector modification resulted in increased MSC survival in vitro and in vivo. Furthermore, decreased apoptosis in the infarcted tissue and improved cardiac function was observed in the Hsp27 group, enhancing the therapeutic effect of MSCs. Together, these data demonstrate that ex vivo genetic modification-specifically Hsp27 overexpression-offers the possibility of enhancing the efficacy of MSC therapy in MI.

Evolution of the Inflammatory and Fibroproliferative Responses during Resolution and Repair after Ventilator-induced Lung Injury in the Rat

Background: The time course and mechanisms of resolution and repair, and the potential for fibrosis following ventilation-induced lung injury (VILI), are unclear. We sought to examine the pattern of inflammation, injury, repair, and fibrosis following VILI. Methods: Sixty anesthetized rats were subject to high-stretch; low-stretch, or sham ventilation, and randomly allocated to undergo periods of recovery of 6, 24, 48, and 96 h, and 7 and 14 days. Animals were then reanesthetized, and the extent of lung injury, inflammation, and repair determined. Results: No injury was seen following low-stretch or sham ventilation. VILI caused severe lung injury, maximal at 24 h, but largely resolved by 96 h. Arterial oxygen tension decreased from a mean (SD) of 144.8 (4.1) mmHg to 96.2 (10.3) mmHg 6 h after VILI, before gradually recovering to 131.2 (14.3) mmHg at 96 h. VILI induced an early neutrophilic alveolitis and a later lymphocytic alveolitis, followed by a monocyte/macrophage infiltration. Alveolar tumor necrosis factor-&agr;, interleukin-1&bgr;, and transforming growth factor-&bgr;1 concentrations peaked at 6 h and returned to baseline within 24 h, while interleukin-10 remained increased for 48 h. VILI generated a marked but transient fibroproliferative response, which restored normal lung architecture. There was no evidence of fibrosis at 7 and 14 days. Conclusions: High-stretch ventilation caused severe lung injury, activating a transient inflammatory and fibroproliferative repair response, which restored normal lung architecture without evidence of fibrosis.

Effects and mechanisms of action of sildenafil citrate in human chorionic arteries

ObjectivesSildenafil citrate, a specific phosphodiesterase-5 inhibitor, is increasingly used for pulmonary hypertension in pregnancy. Sildenafil is also emerging as a potential candidate for the treatment of intra-uterine growth retardation and for premature labor. Its effects in the feto-placental circulation are not known. Our objectives were to determine whether phosphodiesterase-5 is present in the human feto-placental circulation, and to characterize the effects and mechanisms of action of sildenafil citrate in this circulation.Study DesignEx vivo human chorionic plate arterial rings were used in all experiments. The presence of phosphodiesterase-5 in the feto-placental circulation was determined by western blotting and immunohistochemical staining. In a subsequent series of pharmacologic studies, the effects of sildenafil citrate in pre-constricted chorionic plate arterial rings were determined. Additional studies examined the role of cGMP and nitric oxide in mediating the effects of sildenafil.ResultsPhosphodiesterase-5 mRNA and protein was demonstrated in human chorionic plate arteries. Immunohistochemistry demonstrated phosphodiesterase-5 within the arterial muscle layer. Sildenafil citrate produced dose dependent vasodilatation at concentrations at and greater than 10 nM. Both the direct cGMP inhibitor methylene blue and the cGMP-dependent protein kinase inhibitor Rp-8-Br-PET-cGMPS significantly attenuated the vasodilation produced by sildenafil citrate. Inhibition of NO production with L-NAME did not attenuate the vasodilator effects of sildenafil. In contrast, sildenafil citrate significantly enhanced the vasodilation produced by the NO donor sodium nitroprusside.ConclusionPhosphodiesterase-5 is present in the feto-placental circulation. Sildenafil citrate vasodilates the feto-placental circulation via a cGMP dependent mechanism involving increased responsiveness to NO.

Heat Shock Protein 70 Reduces α-Synuclein-Induced Predegenerative Neuronal Dystrophy in the α-Synuclein Viral Gene Transfer Rat Model of Parkinson's Disease

It has become increasingly evident that the nigrostriatal degeneration associated with Parkinsons disease initiates at the level of the axonal terminals in the putamen, and this nigrostriatal terminal dystrophy is either caused or exacerbated by the presence of α‐synuclein immunopositive neuronal inclusions. Therefore, strategies aimed at reducing α‐synuclein‐induced early neuronal dystrophy may slow or halt the progression to overt nigrostriatal neurodegeneration. Thus, this study sought to determine if adeno‐associated virus (AAV) mediated overexpression of two molecular chaperone heat shock proteins, namely Hsp27 or Hsp70, in the AAV‐α‐synuclein viral gene transfer rat model of Parkinsons disease could prevent α‐synuclein‐induced early neuronal pathology.