Mary T. Murray

Signaling of apoptotic lung injury by lipid hydroperoxides.

BACKGROUND Acute lung injury is common after shock and sepsis, but the pathophysiology is unclear. Lipid hydroperoxide products including 4-hydroxynonenal (HNE) increase significantly during these insults and may induce apoptosis. This study investigates the role of pathophysiologic concentrations of HNE on isolated lung biophysical function and apoptosis. METHODS Male Sprague-Dawley rat lungs were isolated and perfused with Krebs-Henseleit buffered solution for 120 minutes. Hydroxynonenal (50 micromol/L) or vehicle was added to the perfusate at 60 minutes. Lung elastance and perfusion pressure were determined. Perfusate glutathione and lactate dehydrogenase were determined at 30-minute intervals. Genomic DNA was extracted for electrophoretic determination of apoptotic laddering. RESULTS There were no differences in any parameter measured before HNE infusion. Lung edema increased significantly with HNE infusion; a trend increase in lung elastance and perfusion pressure was noted. DNA laddering characteristic of apoptosis was noted in HNE-treated lungs that was absent in control animals. CONCLUSION Lipid hydroperoxide products formed during shock or sepsis may be causally related to lung injury. Low concentrations of a candidate metabolite, HNE, appear to induce significant lung injury and apoptosis, which may partially mediate lung injury during shock and sepsis.

Calcium blockade reduces renal apoptosis during ischemia reperfusion

Apoptosis is well described in invertebrates and recently documented in mammals. The prevalence and pathophysiology of mammalian apoptosis is unknown and may have clinical ramifications. The aim of this study is to investigate the apoptotic response during kidney ischemia-reperfusion (I/R) injury. Kidney I/R was initiated in anesthetized rats by occlusion of the renal pedicle for 45 min with or without pretreatment with .2 mg/kg verapamil: control animals received sham exposure. Flow was re-established after ischemia and the animals were allowed to recover for 24 h. Bilateral kidneys were harvested for DNA electrophoresis, Western analysis for p53, Northern analysis for c-myc expression, and light and electron microscopic analysis. Kidney I/R caused characteristic DNA laddering in the clamped kidney, and less extensive laddering was seen in the contralateral kidney. Light and electron microscopic analysis confirmed apoptotic morphology in the reperfused tissues. Verapamil pretreatment completely abolished DNA laddering and attenuated the microscopic evidence of apoptosis. p53 levels were increased by I/R in the ischemic kidney and moderately increased in the contralateral organ, c-myc mRNA levels were increased by the I/R insult. Kidney I/R injury may induce global apoptosis, which seems to be associated with an alteration in calcium homeostasis. The increase in p53 and c-myc mRNA levels seen with I/R may facilitate apoptosis. Calcium modulation seems to reduce apoptosis during I/R and may have therapeutic implications.

In-vivo particle mediated delivery of mRNA to mammalian tissues: Ballistic and biologic effects

Biolistic transmission of mRNA provides transient gene therapy to in vivo organs. This study documents particle mediated mRNA transmission to a solid organ and wound healing model using the mRNA of Green Fluorescent Protein to determine optimal delivery parameters. Renal function, bullet penetration, cellular injury, and Green Fluorescent Protein synthesis were quantified. Chimeric human epidermal growth factor‐FLAG epitope cDNA or mRNA was transmitted to wounds in normal or steroid treated animals. Wound bursting strength, human epidermal growth factor‐FLAG, and collagen synthesis were determined. Injury and bullet penetration correlated with the delivery velocity and bullet size. Optimal delivery parameters were established which provided widespread Green Fluorescent Protein synthesis. Human epidermal growth factor‐FLAG treatment significantly increased collagen content and wound breaking strength in normal and steroid treated animals. FLAG protein synthesis was evident in mRNA treated fascia following treatment. We found the gene gun provides a novel method for efficient, in vivo delivery of mRNA‐based therapeutic strategies to mammalian organs with minimal histologic damage allowing transient expression of protein in in vivo target tissues. Co‐delivery of Green Fluorescent Protein mRNA may provide a useful positive control to determine effective transmission. Biolistic transmission of human epidermal growth factor‐FLAG mRNA provides increased tissue epidermal growth factor levels and accelerates wound healing in normal and steroid exposed animals.