Characterization of endothelial progenitor cells mobilization following cutaneous wounding.

Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are known to play an important role in neovascularization and wound healing. We investigated the temporal effects of cutaneous wounding on EPC surface markers within the peripheral blood and BM, and to better understand the role of the stromal cell-derived factor-1 alpha (SDF-1alpha/CXCR4) axis on EPC mobilization after wounding. FVB/NJ mice were administered bilateral 8 mm circular full-thickness skin wounds. Peripheral blood and BM were isolated at daily intervals postwounding through day 7 and analyzed for EPC mobilization characteristics and levels of SDF-1alpha. Cutaneous wounding was found to cause a transient increase in EPC mobilization that peaked on day 3. In contrast, SDF-1alpha protein within blood plasma was observed to significantly decrease on days 3, 4, and 7 following cutaneous wounding. BM levels of SDF-1alpha protein decreased to a nadir on day 3, the same day as peak mobilization was observed to occur. The decrease in BM SDF-1alpha protein levels was also associated with a decrease in SDF-1alpha mRNA suggesting transcriptional down-regulation as a contributing factor. This study for the first time characterizes EPC mobilization following cutaneous wounding in mice and supports a major role for the SDF-1alpha/CXCR4 axis in regulating mobilization within the BM, without evidence for systemic increases in SDF-1alpha.

Serendipitous discovery of novel bacterial methionine aminopeptidase inhibitors.

In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding.

Intraplacental gene therapy with Ad-IGF-1 corrects naturally occurring rabbit model of intrauterine growth restriction.

Intrauterine growth restriction (IUGR) due to placental insufficiency is a leading cause of perinatal complications for which there is no effective prenatal therapy. We have previously demonstrated that intraplacental injection of adenovirus-mediated insulin-like growth factor-1 (Ad-IGF-1) corrects fetal weight in a murine IUGR model induced by mesenteric uterine artery branch ligation. This study investigated the effect of intraplacental Ad-IGF-1 gene therapy in a rabbit model of naturally occurring IUGR (runt) due to placental insufficiency, which is similar to the human IUGR condition with onset in the early third trimester, brain sparing, and a reduction in liver weight. Laparotomy was performed on New Zealand White rabbits on day 21 of 30 days of gestation and litters were divided into five groups: Control (first position)+phosphate-buffered saline (PBS), control+Ad-IGF-1, runt (third position)+PBS, runt+Ad-IGF-1, and runt+Ad-LacZ. The effect of IGF-1 gene therapy on fetal, placental, liver, heart, lung, and musculoskeletal weights of the growth-restricted pups was examined. Protein expression after gene transfer was seen along the maternal-fetal placenta interface (n=12) 48 hr after gene therapy. There was minimal gene transfer detected in the pups or maternal organs. At term, compared with the normally grown first-position control, the runted third-position pups demonstrated significantly lower fetal, placental, liver, lung, and musculoskeletal weights. The fetal, liver, and musculoskeletal weights were restored to normal by intraplacental Ad-IGF-1 gene therapy (p<0.01), with no change in the placental weight. Intraplacental gene therapy is a novel strategy for the treatment of IUGR caused by placental insufficiency that takes advantage of an organ that will be discarded at birth. Development of nonviral IGF-1 gene delivery using placenta-specific promoters can potentially minimize toxicity to the mother and fetus and facilitate clinical translation of this novel therapy.

Interaction between human placental microvascular endothelial cells and a model of human trophoblasts: effects on growth cycle and angiogenic profile.

Abstract Intrauterine growth restriction (IUGR) is a leading cause of perinatal complications, and is commonly associated with reduced placental vasculature. Recent studies demonstrated over-expression of IGF-1 in IUGR animal models maintains placental vasculature. However, the cellular environment of the placental chorionic villous is unknown. The close proximity of trophoblasts and microvascular endothelial cells in vivo alludes to autocrine/paracrine regulation following Ad-HuIGF-1 treatment. We investigated the co-culturing of BeWo Choriocarcinoma and Human Placental Microvascular Endothelial Cells (HPMVECs) on the endothelial angiogenic profile and the effect Ad-HuIGF-1 treatment of one cell has on the other. HPMVECs were isolated from human term placentas and cultured in EGM-2 at 37°C with 5% CO2. BeWo cells were maintained in Ham's F12 nutrient mix with 10% FBS and 1% pen/strep. Co-cultured HPMVECS+BeWo cells were incubated in serum-free control media, Ad-HuIGF-1, or Ad-LacZ at MOI 0 and MOI 100:1 for 48 h. Non-treated cells and mono-cultured cells were compared to co-cultured cells. Angiogenic gene expression and proliferative and apoptotic protein expression were analysed by RT-qPCR and immunocytochemistry, respectively. Statistical analyses was performed using student's t-test with P < 0.05 considered significant. Direct Ad-HuIGF-1 treatment increased HPMVEC proliferation (n = 4) and reduced apoptosis (n = 3). Co-culturing HPMVECs+BeWo cells significantly altered RNA expression of the angiogenic profile compared to mono-cultured HPMVECs (n = 8). Direct Ad-HuIGF-1 treatment significantly increased Ang-1 (n = 4) in BeWo cells. Ad-HuIGF-1 treatment of HPMVECs did not alter the RNA expression of angiogenic factors. Trophoblastic factors may play a key role in placental vascular development and IGF-1 may have an important role in HPMVEC growth.

Compensatory lung growth in NOS3 knockout mice suggests synthase isoform redundancy.

Nitric oxide synthase 3 (NOS3) produces nitric oxide (NO) in endothelial cells, which stimulates cyclic guanosine monophosphate (cGMP) production and thereby mediates pulmonary vasodilation. Inhibition of cGMP enzymatic cleavage by sildenafil might be involved in lung growth stimulating processes in pulmonary hypoplasia. The aim of this study was to discover insights into the transcriptional regulation of NOS3 in a mouse model of compensatory lung growth (CLG). CLG was studied in wild type animals (WT) and NOS3 knockout mice (NOS3-/-) by dry weight, DNA, and protein quantification as well as relative quantification of NOS mRNA. All assessments were done on adult female mice, 10 days after left pneumonectomy (PNX) or sham thoracotomy. Weight ratios of right NOS3-/- lungs were no different than controls. There was a compensatory increase in DNA and a noncompensating increase in protein ratios in NOS3-/- mice compared with controls. Pharmacological knockdown with the pan-NOS inhibitor l-NAME (nitro-arginine methyl ester) reduced CLG by only 8% compared with the d-NAME treated control mice. Relative quantification of lung mRNA revealed no up-regulation of NOS3 expression in WT lungs after PNX, but NOS3-/- lungs showed a 2.6-fold higher inducible NOS2 expression compared with shams. These data suggest that NOS3 loss of function alone does not impair CLG in mice, possibly because of redundancy mechanisms involving NOS2.