The EXTrauterine Environment for Neonatal Development Supports Normal Intestinal Maturation and Development.

BACKGROUND AND AIMS: The Extra-Uterine Environment for Neonatal Development (EXTEND) aims to avoid the complications of prematurity, such as NEC. Our goal was to determine if bowel development occurs normally in EXTEND-supported lambs, with specific emphasis on markers of immaturity associated with NEC. METHODS: We compared terminal ileum from 17 pre-term lambs supported on EXTEND for 2- 4 weeks to bowel from age-matched fetal lambs that developed in utero. We evaluated morphology, markers of epithelial integrity and maturation, enteric nervous system structure, and bowel motility. RESULTS: EXTEND-supported lamb ileum had normal villus height, crypt depth, density of mucin-containing goblet cells, and enteric neuron density. Expression patterns for I-FABP, activated caspase-3 and EGFR were normal in bowel epithelium. Transmural resistance assessed in Ussing chambers was normal. Bowel motility was also normal as assessed by ex vivo organ bath and video imaging. However, Peyer's patch organization did not occur normally in EXTEND ileum, resulting in fewer circulating B cells in experimental animals. CONCLUSION: EXTEND supports normal ileal epithelial and enteric nervous system maturation in pre-term lambs. The classic morphologic changes and cellular expression profiles associated with NEC are not seen. However, immune development within the EXTEND supported lamb bowel does not progress normally.

Stable long-term mixed chimerism achieved in a canine model of allogeneic in utero hematopoietic cell transplantation.

Evidence supporting the efficacy of in utero hematopoietic cell transplantation (IUHCT) in a valid large animal model is needed prior to clinical application. The objective of this study was to establish clinically relevant levels of hematopoietic chimerism in a canine model of maternal-to-fetal IUHCT. We first assessed immune and hematopoietic ontogeny relevant to IUHCT in the canine model and identified 40 days' gestation (term 63 days) as a time point at the initiation of thymic selection, and prior to bone marrow hematopoiesis, that might be optimal for IUHCT. We next determined that intravascular administration of donor cells via intracardiac injection was far more efficient and resulted in much higher levels of donor cell engraftment than intraperitoneal injection. By applying these findings, we achieved stable long-term multilineage engraftment in 21 of 24 surviving recipients with an average level of initial chimerism of 11.7% (range 3% to 39%) without conditioning or evidence of graft-versus-host disease. Donor cell chimerism remained stable for up to 2 years and was associated with donor-specific tolerance for renal transplantation. The levels of donor cell chimerism achieved in this study would be therapeutic for many hematopoietic disorders and are supportive of a clinical trial of IUHCT.

In utero lung gene transfer using adeno-associated viral and lentiviral vectors in mice.

Virus-mediated gene transfer to the fetal lung epithelium holds considerable promise for the therapeutic management of prenatally diagnosed, potentially life-threatening inherited lung diseases. In this study we hypothesized that efficient and life-long lung transduction can be achieved by in utero gene therapy, using viral vectors. To facilitate diffuse entry into the lung, viral vector was injected into the amniotic sac of C57BL/6 mice on embryonic day 16 (term, ∼ 20 days) in a volume of 10 µl. Vectors investigated included those based on adeno-associated virus (AAV) (serotypes 5, 6.2, 9, rh.64R1) and vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1-based lentivirus (LV). All vectors expressed green fluorescent protein (GFP) under the transcriptional control of various promoters including chicken ß-actin (CB) or cytomegalovirus (CMV) for AAV and CMV or MND (myeloproliferative sarcoma virus enhancer, negative control region deleted) for LV. Pulmonary GFP gene expression was detected by fluorescence stereoscopic microscopy and immunohistochemistry for up to 9 months after birth. At equivalent vector doses (mean, 12 × 10(10) genome copies per fetus) three AAV vectors resulted in long-term (up to 9 months) pulmonary epithelium transduction. AAV2/6.2 transduced predominantly cells of the conducting airway epithelium, although transduction decreased 2 months after vector delivery. AAV2/9-transduced cells of the alveolar epithelium with a type 1 pneumocyte phenotype for up to 6 months. Although minimal levels of GFP expression were observed with AAV2/5 up to 9 months, the transduced cells immunostained positive for F480 and were retrievable by bronchoalveolar lavage, confirming an alveolar macrophage phenotype. No GFP expression was observed in lung epithelial cells after AAV2/rh.64R1 and VSV-G-LV vector-mediated gene transfer. We conclude that these experiments demonstrate that prenatal lung gene transfer with AAV vectors engineered to target pulmonary epithelial cells may provide sustained long-term levels of transgene expression, supporting the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

Lentiviral-mediated over-expression of hyaluronan synthase-1 (HAS-1) decreases the cellular inflammatory response and results in regenerative wound repair.

Fetal wounds have been found to have increased levels of high-molecular-weight hyaluronan (HMW-HA) compared with those of adults. The primary enzyme responsible for producing HMW-HA is hyaluronic acid synthase-1 (HAS-1). We hypothesized that over-expression of HAS-1 in adult dermal wounds would decrease inflammation and promote regenerative healing. To test this hypothesis, the flanks of adult C57Bl/6 mice were treated with a lentiviral construct containing either HAS-1-GFP or GFP transgenes. After 48 h, a 4-mm excisional wound was made at the site of treatment. Wounds were harvested at days 3, 7, or 28 after wounding. Wound phenotype was assessed by histology to examine tissue architecture and immunohistochemistry for CD45. At 7 and 28 days, lenti-HAS-1-treated wounds demonstrated the restoration of the normal dermal elements and organized collagen fiber orientation. In contrast, the lenti-GFP-treated wounds lacked normal dermal architecture and demonstrated a disorganized collagen scar. At 3 and 7 days, wounds treated with lenti-HAS-1 exhibited a significant decrease in the number of inflammatory cells when compared with wounds treated with lenti-GFP. Thus, HAS-1 over-expression promotes dermal regeneration, in part by decreasing the inflammatory response and by recapitulation of fetal extracellular matrix HMW-HA content.

Amniotic fluid levels of glial fibrillary acidic protein in fetal rats with retinoic acid induced myelomeningocele: a potential marker for spinal cord injury.

OBJECTIVE: The objective of this study was to determine whether amniotic fluid levels of glial acidic fibrillary protein (GFAP) would reflect myelomeningocele-related neurodegeneration in the rat model of retinoic acid-induced myelomeningocele, which is a model that is very similar to human myelomeningocele and develops the entire spectrum of disease severity including features of the Chiari II malformation. STUDY DESIGN: Time-dated (embryonic day 10) pregnant Sprague-Dawley rats were gavage fed 60 mg/kg/bodyweight retinoic acid that had been dissolved in olive oil or olive oil alone. Myelomeningocele, retinoic acid-exposed no myelomeningocele, and control fetuses were harvested at specific time points throughout gestation. A standard set of pinching tests was performed to interrogate the sensorimotor reflex arc of hindpaws and tails. Amniotic fluid-GFAP levels were analyzed by standard enzyme-linked immunosorbent assay techniques. RESULTS: Amniotic fluid-GFAP levels were similar between groups at embryonic days 14, 16, and 18, respectively. Compared with control fetuses, amniotic fluid GFAP levels were significantly increased in myelomeningocele fetuses at embryonic days 20 and 22 (P < .001). Defect size (P < .001), presence of clubfoot deformity (P = .0004), and absence of sensorimotor function (P < .01) at embryonic day 22 correlated with amniotic fluid-GFAP levels. CONCLUSION: Amniotic fluid-GFAP levels appear to correlate with spinal cord injury as gestation proceeds in fetal rats with myelomeningocele.

Inhibition of stromal cell-derived factor-1α further impairs diabetic wound healing.

OBJECTIVE: Impaired diabetic wound healing is associated with abnormal stromal cell-derived factor (SDF)-1α production, decreased angiogenesis, and chronic inflammation. Lentiviral-mediated overexpression of SDF-1α can correct the impairments in angiogenesis and healing in diabetic wounds. We hypothesized that SDF-1α is a critical component of the normal wound-healing response and that inhibition of SDF-1α would further delay the wound-healing process. METHODS: dB/Db diabetic mice and Db/+ nondiabetic mice were wounded with an 8-mm punch biopsy and the wounds treated with a lentiviral vector containing either the green fluorescent protein (GFP) or SDF-1α inhibitor transgene. The inhibitor transgene is a mutant form of SDF-1α that binds, but does not activate, the CXCR4 receptor. Computerized planimetry was used to measure wound size daily. Wounds were analyzed at 3 and 7 days by histology and for production of inflammatory markers using real-time polymerase chain reaction. The effect of the SDF-1α inhibitor on cellular migration was also assessed. RESULTS: Inhibition of SDF-1α resulted in a significant decrease in the rate of diabetic wound healing, (3.8 vs 6.5 cm(2)/day in GFP-treated wounds; P = .04), and also impaired the early phase of nondiabetic wound healing. SDF-1α inhibition resulted in fewer small-caliber vessels, less granulation tissue formation, and increased proinflammatory gene expression of interleukin-6 and macrophage inflammatory protein-2 in the diabetic wounds. CONCLUSIONS: The relative level of SDF-1α in the wound plays a key role in the wound-healing response. Alterations in the wound level of SDF-1α, as seen in diabetes or by SDF-1α inhibition, impair healing by decreasing cellular migration and angiogenesis, leading to increased production of inflammatory cytokines and inflammation. Inhibition of SDF-1α further impairs diabetic wound healing.

Fetal tendon wound size modulates wound gene expression and subsequent wound phenotype.

The fetal response to small tendon injury results in regenerative or scarless healing and is characterized by a markedly diminished cellular inflammatory response, lack of fibroplasia, and restoration of normal tissue architecture. We hypothesized that an increasing fetal tendon wound size would lead to increased wound inflammation and a change from regenerative to reparative healing and scar formation. We created small or large tendon wounds in early gestation fetal sheep and used histology to assess tissue architecture, immunohistochemistry to assess the cellular inflammatory response, ovine-specific gene microarrays, and real-time reverse transcription-polymerase chain reaction to measure the gene expression in response to injury. Small tendon wounds showed a regenerative healing phenotype with orderly deposition of collagen fibers while large tendon wounds showed disorderly collagen deposition consistent with scar formation. Small tendon wounds had few inflammatory cells at 7 and 28 days after injury, whereas the large wounds showed a significant inflammatory cell infiltrate at 7 days that resolved by 28 days. At 3 days, the differential expression of genes involved in the response to injury and inflammation were seen between large and small tendon wounds. By real-time polymerase chain reaction at 7 days, large tendon wounds also had significantly increased expression of interleukin-6, interleukin-8, transforming growth factor-ß1, and transforming growth factor-ß3, compared with the small wounds. Increasing the fetal tendon wound size results in increased proinflammatory gene expression, inflammatory cell infiltration, and a change from regenerative to reparative healing. This model allows the process of regenerative healing to be examined without the confounding variable of gestational age.

Gastroschisis in mice lacking aortic carboxypeptidase-like protein is associated with a defect in neuromuscular development of the eviscerated intestine.

Mice lacking aortic carboxypeptidase-like protein (ACLP) exhibit a gastroschisis (GS) like abdominal wall defect. The objectives of this study were to evaluate the pathophysiological features of GS in ACLP mice and to characterize the neuromuscular development of the eviscerated intestine (EI). ACLP mice were created by heterozygous mating from previously generated mice with targeted disruption of ACLP. Specimens were processed for H&E, and immunohistochemistry for smooth muscle cells [SMC, alpha-smooth muscle actin (alpha-SMA) antibody], interstitial cells of Cajal (ICC, c-kit-antibody), neural crest cells (NCC, Hox-b5-antibody), and enteric neurons (EN, PGP9.5-, alpha-internexin, and synaptophysin antibody). From 47 fetuses genotyped, 13 (27.7%) were wild type, 20 (42.5%) were heterozygous, and 14 (29.8%) were ACLP homozygous. In GS mice, expression of c-kit, Hox-b5, PGP-9.5, alpha-internexin, and synaptophysin were almost completely absent and only faint alpha-SMA expression was seen in the EI. In contrast, c-kit, Hox-b5, PGP9.5, alpha-internexin, synaptophysin, and alpha-SMA expression in intra-abdominal intestine in GS fetuses was the same as control intestine. The defect observed in ACLP mice closely resembles GS. Absence of ICC, NCC, EN, and immature differentiation of SMC supports an associated defect in neuromuscular development that is restricted to the EI.

Safety of AAV factor IX peripheral transvenular gene delivery to muscle in hemophilia B dogs.

Muscle represents an attractive target tissue for adeno-associated virus (AAV) vector-mediated gene transfer for hemophilia B (HB). Experience with direct intramuscular (i.m.) administration of AAV vectors in humans showed that the approach is safe but fails to achieve therapeutic efficacy. Here, we present a careful evaluation of the safety profile (vector, transgene, and administration procedure) of peripheral transvenular administration of AAV-canine factor IX (cFIX) vectors to the muscle of HB dogs. Vector administration resulted in sustained therapeutic levels of cFIX expression. Although all animals developed a robust antibody response to the AAV capsid, no T-cell responses to the capsid antigen were detected by interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISpot). Interleukin (IL)-10 ELISpot screening of lymphocytes showed reactivity to cFIX-derived peptides, and restimulation of T cells in vitro in the presence of the identified cFIX epitopes resulted in the expansion of CD4(+)FoxP3(+)IL-10(+) T-cells. Vector administration was not associated with systemic inflammation, and vector spread to nontarget tissues was minimal. At the local level, limited levels of cell infiltrates were detected when the vector was administered intravascularly. In summary, this study in a large animal model of HB demonstrates that therapeutic levels of gene transfer can be safely achieved using a novel route of intravascular gene transfer to muscle.

Correction of murine ADAMTS13 deficiency by hematopoietic progenitor cell-mediated gene therapy.

ADAMTS13, a metalloprotease primarily synthesized in liver and endothelial cells, cleaves von Willebrand factor (VWF) at the central A2 domain, thereby reducing the sizes of circulating VWF multimers. Genetic or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). To date, plasma infusion or exchange is the only proven effective therapy for TTP. In search for a better therapy, an autologous transplantation of hematopoietic progenitor cells transduced ex vivo with a self-inactivating lentiviral vector encoding a full-length murine Adamts13 and an enhanced green fluorescent protein (GFP) reporter gene was performed in Adamts13(-/-) mice after irradiation. All recipient mice showed detectable ADAMTS13 antigen and proteolytic activity in plasma despite only low levels of bone marrow chimerism. The levels of plasma ADAMTS13 were sufficient to eliminate the ultralarge VWF multimers and offered systemic protection against ferric chloride-induced arterial thrombosis. The data suggest that hematopoietic progenitor cells can be genetically modified ex vivo and transplanted in an autologous model to provide adequate levels of functional ADAMTS13 metalloprotease. This success may provide the basis for development of a novel therapeutic strategy to cure hereditary TTP in humans.

IL-10 overexpression decreases inflammatory mediators and promotes regenerative healing in an adult model of scar formation.

Adult wound healing is characterized by an exuberant inflammatory response and scar formation. In contrast, scarless fetal wound healing has diminished inflammation, a lack of fibroplasia, and restoration of normal architecture. We have previously shown that fetal wounds produce less inflammatory cytokines, and the absence of IL-10, an anti-inflammatory cytokine, results in fetal scar formation. We hypothesized that increased IL-10 would decrease inflammation and create an environment conducive for regenerative healing in the adult. To test this hypothesis, a lentiviral vector expressing IL-10 and green fluorescent protein (GFP) (Lenti-IL-10) or GFP alone (Lenti-GFP) was injected at the wound site 48 hours before wounding. We found that both Lenti-IL-10 and Lenti-GFP were expressed in the wounds at 1 and 3 days post wounding. At 3 days, Lenti-IL-10-treated wounds demonstrated decreased inflammation and decreased quantities of all proinflammatory mediators analyzed with statistically different levels of IL-6, monocyte chemoattractant protein-1, and heat-shock protein 47. At 3 weeks, Lenti-GFP wounds demonstrated scar formation. In contrast, wounds injected with Lenti-IL-10 demonstrated decreased inflammation, a lack of abnormal collagen deposition, and restoration of normal dermal architecture. We conclude that lentivirus-mediated overexpression of IL-10 decreases the inflammatory response to injury, creating an environment conducive for regenerative adult wound healing.

Permissive environment in postnatal wounds induced by adenoviral-mediated overexpression of the anti-inflammatory cytokine interleukin-10 prevents scar formation.

Wound healing in the mid-gestation fetus is scarless with minimal inflammation and a unique extracellular matrix. We have previously documented the relative lack of inflammatory cytokines in this environment. We demonstrate that interleukin (IL)-10 is highly expressed in mid-gestation human fetal skin but is absent in postnatal human skin. We hypothesize that overexpression of IL-10 in postnatal skin may replicate a permissive environment for scarless healing. To study the mechanism underlying this process we performed immunohistochemistry for IL-10 in human mid-gestation fetal and postnatal skin. We also determined if adenoviral-mediated overexpression of IL-10 could allow for scarless wound healing in a murine incisional wound model. Wounds were analyzed at 1-90 days postwounding for effects on scar formation, inflammatory response, and biomechanical properties. Ad-IL-10 reconstitutes a permissive environment for scarless healing as shown by reconstitution of a normal dermal reticular collagen pattern and distribution of dermal elements. Compared with controls, Ad-IL-10 treated wounds showed reduced inflammatory response and no difference in biomechanical parameters. Therefore, overexpression of IL-10 in postnatal wounds results in a permissive environment for scarless wound repair, possibly by replicating a fetal wound environment.

Morphologic analysis of the neuromuscular development of the anorectal unit in fetal rats with retinoic acid induced myelomeningocele.

To investigate whether myelomeningocele (MMC) is associated with a global neuromuscular maldevelopment of the lower gastrointestinal (GI) tract and anorectum, the distribution and staining intensity of non-neuronal (alpha-smooth-muscle-actin), neural crest cell (NCC, [Hoxb5]), and neuronal markers (PGP-9.5, synaptophysin, neurotubulin-beta-III) within the distal colon, rectum, and anal sphincters were analyzed by immunohistochemistry and Western blot in rat fetuses with retinoic acid (RA) induced MMC. At term (E22), no gross-morphological differences of the anorectal unit of OIL (n=21) MMC (n=31), and RA-exposed-non MMC (RA, n=19) fetuses were found. Smooth muscle cells were evenly distributed within the muscle layers of the rectum and the internal anal sphincter in OIL, MMC, and RA fetuses. Density and staining intensity of NCC and mature enteric neurons within the myenteric plexus of the distal colon and rectum and innervation pattern within anal sphincters in MMC fetuses were analogous to RA and OIL controls. Normal smooth muscle and myenteric plexus development of the rectum and normal innervation of the anal sphincters and pelvic floor suggests that MMC is not associated with a global neuromuscular maldevelopment of lower GI structures in this short-gestational model.

Efficient in vivo targeting of epidermal stem cells by early gestational intraamniotic injection of lentiviral vector driven by the keratin 5 promoter.

At the present time, no efficient in vivo method for gene transfer to skin stem cells exists. In this study, we hypothesized that early in gestation, specific epidermal stem cell populations may be accessible for gene transfer. To test this hypothesis, we injected lentiviral vectors encoding the green fluorescence protein marker gene driven by either the cytomegalovirus promoter or the keratin 5 (K5) promoter into the murine amniotic space at early developmental stages between embryonic days 8 and 12. This resulted in sustained green fluorescent protein (GFP) expression in both basal epidermal stem cells and bulge cells in the hair follicles of the skin. Transduction of stem cell populations was dependent on the developmental stage, and confirmed by the prolonged duration of GFP expression in all skin elements into adulthood. In addition, transduced stem cell populations responded to regenerative signals after wounding and actively participated in wound healing. Finally, we quantified the fraction of epidermal stem cells transduced, and the distribution of transduction related to the promoters utilized, confirming improved efficiency with the K5 promoter. This simple approach has possible biological applications in our study of gene functions in skin, and perhaps future clinical applications for treatment of skin based disorders.

Stromal progenitor cell therapy corrects the wound-healing defect in the ischemic rabbit ear model of chronic wound repair.

Chronic wounds create a formidable clinical problem resulting in considerable morbidity and healthcare expenditure. The etiology for wound healing impairment appears to be multifactorial; however, ischemia is a common factor in most types of chronic wounds. Ideal therapy for such wounds would be to correct deficiencies in growth factors and matrix components and provide cellular precursors required for timely wound closure. We hypothesized that stromal progenitor cell (SPC) therapy could correct the ischemic wound-healing defect through both direct and indirect mechanisms. To test this hypothesis, we used the ischemic rabbit ear model of chronic wound healing. We found that treatment of the wounds with SPCs was able to reverse the ischemic wound-healing impairment, with improved granulation tissue formation and reepithelialization compared with vehicle or bone marrow mononuclear cell controls. In vitro, SPCs were found to produce factors involved in angiogenesis and reepithelialization, and extracellular matrix components, providing evidence for both direct and indirect mechanisms for the observed correction of the healing impairment in these wounds. Treatment of ischemic wounds with SPCs can dramatically improve wound healing and provides a rationale for further studies focused on SPCs as a potential cellular therapy in impaired wound healing.

Enhanced epithelial gap closure and increased angiogenesis in wounds of diabetic mice treated with adult murine bone marrow stromal progenitor cells.

The direct application of bone marrow (BM) can accelerate the healing of chronic wounds. We hypothesized that this effect is due to the presence of stromal progenitor cells (SPCs) found within whole BM preparations. To test this hypothesis, we isolated adult murine SPCs from whole BM and examined their ability to enhance impaired wound healing compared with ficoll separated BM cells in the diabetic (db/db) mouse model. SPCs significantly enhanced reepithelialization, granulation tissue formation, and neovascularization compared with control wounds treated with BM or PBS alone. Higher frequencies of donor SPC cells compared with donor BM cells were observed in treated wounds at 7 days. Transdifferentiation into GFP-positive mature endothelial cells was not observed. These observations suggest that SPCs improve wound healing through indirect mechanisms which lead to enhanced vascularization rather than through direct participation and incorporation into tissue. We conclude that topical application of BM-derived SPCs may represent an effective strategy for the treatment of chronic diabetic wounds.

Gene transfer to ocular stem cells by early gestational intraamniotic injection of lentiviral vector.

Ocular gene transfer has generally been approached by direct intraocular injection. In this study, we hypothesized that an opportunity exists during early gestation when specific ocular stem cell populations are accessible for gene transfer. These include the stem cell populations that maintain the cornea, lens, and retina throughout life. To test this hypothesis, we injected lentiviral vector encoding the green fluorescent protein (GFP) reporter gene into the murine amniotic space from the late head fold/early somite stage postcoital day 8 (E8) to E18 and performed sequential analysis of GFP expression in ocular tissues. Depending on the timing of vector exposure, significant GFP expression was observed in all ectoderm-derived tissues in the eye. With injection at early gestational time points, GFP expression persisted long term, with evidence of high efficiency stem cell transduction in the cornea, lens, and retina. The observed patterns and duration of gene expression confirm the accessibility of ocular stem cell populations for lentiviral vector-based gene transfer at specific developmental time points in early gestation. This model may be useful for the investigation of mechanisms of genetic and/or developmental ocular disease and for the development of prenatal gene therapy for specific ocular disorders.

Structural and functional characterization of bladder smooth muscle in fetal rats with retinoic acid-induced myelomeningocele.

Myelomeningocele (MMC) is the most common cause of neurogenic bladder dysfunction (NBD). We recently developed a novel retinoic acid (RA)-induced MMC model in fetal rats. The objective of this study was to use this model to assess functional and structural characteristics of the detrusor muscle in MMC-associated NBD. Time-dated pregnant Sprague-Dawley rats were gavage fed 60 mg/kg RA dissolved in olive oil or olive oil alone [embryonic day 10 (E10)]. Bladder specimens from olive oil-exposed fetuses (OIL; n = 71), MMC (n = 79), and RA-exposed-no MMC (RA, n = 62) were randomly assigned for functional and histopathological evaluation and protein analysis. Contractility responses to field and agonist-mediated stimulation (KCl and bethanecol) were analyzed. The expression patterns of alpha-smooth muscle actin, myosin, desmin, vimentin, and collagen III and I were analyzed by immunohistochemistry and Western blotting. Spatial and temporal distribution of nerve fibers within the detrusor muscle was monitored by neurotubulin-beta-III throughout gestation. Neither OIL, MMC, nor RA detrusor responded to field stimulation. MMC bladder strips showed a significant decrease in contractility after KCl and bethanechol stimulation compared with OIL and RA bladders. Bladder detrusor morphology and expression patterns of smooth muscle markers were similar between groups. Detrusor muscles in OIL and RA fetuses were densely innervated, possessing abundant intramural ganglia and nerve trunks that branch to supply smooth muscle bundles. In MMC bladders, neurotubulin-beta-III-positive nerve fibers were markedly decreased with advancing gestational age and were almost completely absent at term (E22). We conclude that the biomechanical properties of fetal rat MMC bladders are analogous to that seen in humans with MMC-associated NBD. Decreased nerve density indicates loss of peripheral neural innervation throughout gestation. The early observation of decreased innervation and decreased contractility in the absence of morphologic abnormalities in muscle structure or extracellular matrix supports a pathophysiological hypothesis that denervation is the primary insult preceding the observed alterations in bladder muscle structure and function.

Transduction of satellite cells after prenatal intramuscular administration of lentiviral vectors.

BACKGROUND: We have previously reported long-term expression of lacZ in myocytes after in utero intramuscular injection of Mokola and Ebola pseudotyped lentiviral vectors. In further experiments, we have noted that these vectors also transduce small cells at the periphery of the muscle fibers that have the morphology of satellite cells, or muscle stem cells. In this study we performed experiments to further define the morphology and function of these cells. METHODS: Balb/c mice at 14-15 days gestation were injected intramuscularly with Ebola or Mokola pseudotyped lentiviral vectors carrying CMV-lacZ. Animals were harvested at various time points, muscles were stained with X-gal, and processed for electron microscopy (EM) and immunofluorescence. To determine whether transduced satellite cells were functionally capable of regenerating injured muscles, animals were injected with notexin in the same area 8 weeks after the in utero injection of viral vector. RESULTS: Transmission EM of transduced cells confirmed the ultrastructural appearance of satellite cells. Double immunofluorescence for beta-galactosidase and satellite cell markers demonstrated co-localization of these markers in transduced cells. In the notexin-injured animals, small blue cells were seen at the areas of regeneration that co-localized beta-galactosidase with markers of regenerating satellite cells. Central nucleated blue fibers were seen at late time points, indicating regenerated muscle fibers arising from a transduced satellite cell. CONCLUSIONS: This study demonstrates transduction of muscle satellite cells following prenatal viral vector mediated gene transfer. These findings may have important implications for gene therapy strategies directed toward muscular dystrophy.

Adenoviral mediated gene transfer of PDGF-B enhances wound healing in type I and type II diabetic wounds.

We have shown that the genetically diabetic mouse (C57BLKS/J-m+/+Lepr(db)) has a wound healing and neovascularization deficit associated with an inability to recruit endothelial precursor cells (EPCs) to the wound. This may account for a fundamental mechanism in impaired diabetic wound healing. We hypothesized that the adenoviral mediated overexpression of platelet-derived growth factor-B (PDGF-B) would enhance wound healing, improve neovascularization, and recruit EPCs to the epithelial wound in three diabetic mouse models. Eight-mm full-thickness flank wounds were made in db/db, nonobese NOD/Ltj, streptozotocin, and C57BLKS/J mice. Wounds were treated with either 1 x 10(8) PFU Ad-PDGF-B or Ad LacZ or phosphate buffered saline solution. Wounds harvested at seven days were analyzed for epithelial gap, blood vessel density, granulation tissue area, and EPCs per high powered field. All three diabetic models have a significant wound healing and neovascularization defect compared to C57BLKS/J controls. Adenoviral-PDGF-B treatment significantly enhanced epithelial gap closure in db/db, streptozotocin, and nonobese NOD/Ltj mice as compared to diabetic phosphate buffered saline solution or Ad LacZ controls. A similar increase in the formation of granulation tissue and vessel density was also observed. All three models had reduced levels of GATA-2 positive EPCs in the wound bed that was corrected by the adenoviral mediated gene transfer of PDGF. EPC recruitment was positively correlated with neovascularization and wound healing. Three different diabetic models have a wound healing impairment and a decreased ability to recruit EPCs. The vulnerary effect of adenoviral mediated gene therapy with PDGF-B significantly enhanced wound healing and neovascularization in diabetic wounds. The PDGF-B mediated augmentation of EPC recruitment to the wound bed may be a fundamental mechanism of these results.