Herpes simplex virus mediated nerve growth factor expression in bladder and afferent neurons: potential treatment for diabetic bladder dysfunction.

PURPOSE: Diabetic cystopathy resulting from sensory neuropathy may potentially be treated by direct gene therapy. It has been suggested that nerve growth factor (NGF) has an ameliorative effect in preventing the death in diabetes of afferent dorsal root ganglion neurons, which control bladder function. We investigated NGF gene transfer to the bladder and bladder afferent pathways for treating diabetic cystopathy. We used replication competent and replication defective herpes simplex virus type 1 (HSV-1) vectors that express a functionally active form of the beta-subunit of mouse NGF (beta-NGF) to examine the level and duration of therapeutic gene expression after administration of the vectors. MATERIALS AND METHODS: NGF expression during acute (3 days) and latent (21 days) infections was assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical testing after the injection of 1 x 106 to 1 x 108 pfu HSV-NGF expression vectors into the bladder wall of adult rats. RESULTS: HSV vectors with the strong human cytomegalovirus immediate early promoter used to drive beta-NGF gene expression exhibited increased NGF 3 days after infection in the bladder and L6 to S1 dorsal root ganglia, where bladder afferent neurons are located. ELISA analysis revealed that NGF in the bladder tissue and dorsal root ganglia was increased 7 to 9 and 2 to 4-fold, respectively, over the control vector. Increased NGF expression in L6 to S1 dorsal root ganglia neurons was also detected by immunohistochemical staining with antiNGF antibodies. Extended NGF expression was detected by ELISA 21 days after injection. Replication defective vectors containing HSV-1 latency promoter (LAP-2) driving NGF expressed NGF in the bladder and dorsal root ganglia 21 days after bladder injection. ELISA analysis confirmed an approximate 2 to 3-fold increase of NGF expression in the bladder and L6 to S1 dorsal root ganglia. CONCLUSIONS: The NGF gene may be transferred and expressed in the bladder and bladder afferent pathways using HSV vectors. To our knowledge our study represents the first demonstration of the effectiveness of gene therapy for altering neurotrophic expression in visceral sensory neurons. This technique of gene transfer may be useful for treating certain types of neurogenic bladder dysfunction, such as diabetic cystopathy, in which decreased NGF transport may be a causative factor.

Botulinum toxin urethral sphincter injection to restore bladder emptying in men and women with voiding dysfunction.

PURPOSE: Botulinum toxin injection into the external urinary sphincter in spinal cord injured men with detrusor-sphincter dyssynergia has been reported. We expand the clinical use of botulinum toxin for a variety of bladder outlet obstructions and to decrease outlet resistance in patients with acontractile detrusor but who wish to void by the Valsalva maneuver. MATERIALS AND METHODS: Prospective treatment was performed for voiding dysfunction in 8 men and 13 women 34 to 74 years old. The reasons for voiding dysfunction included neurogenic detrusor-sphincter dyssynergia in 12 cases, pelvic floor spasticity in 8 and acontractile detrusor in 1 patient with multiple sclerosis who wished to void by the Valsalva maneuver. Using a rigid cystoscope and a collagen injection needle, a total of 80 to 100 units of botulinum A toxin (Botox) were injected into the external sphincter at the 3, 6, 9 and 12 o'clock positions. RESULTS: Preoperatively 19 of 21 patients were on indwelling or intermittent catheterization. After botulinum A injection all but 1 patient were able to void without catheterization. No acute complications, such as general paralysis or respiratory depression, occurred and none of the patients had dribbling or stress urinary incontinence. Postoperative post-void residual decreased by 71% and voiding pressures decreased on average 38%. Of the 21 patients 14 (67%) reported significant subjective improvement in voiding. Followup ranges from 3 to 16 months, with a maximum of 3 botulinum A injections in some patients. CONCLUSIONS: Urethral sphincter botulinum injection should be considered for complex voiding dysfunction. Encouraging improvement without complications were seen in most of our patients. We have expanded the use of botulinum toxin to treat pelvic floor spasticity and also women.

Urolume stent placement for the treatment of postbrachytherapy bladder outlet obstruction.

OBJECTIVES: Transurethral resection (TURP) or incision of the prostate is generally not effective for treating bladder outlet obstruction after transperineal brachytherapy for prostate cancer. Furthermore, TURP could compromise full-dose effective radiation delivery to the prostate. We analyzed the efficacy of the UroLume stent in treating the urinary outflow obstruction in such patients. METHODS: Five patients who had undergone brachytherapy (3 with (192)Ir high-dose radiation and 2 with (125)I) subsequently developed one or more episodes of urinary retention 2 weeks to 4 years after treatment. The patients failed or could not tolerate alpha-blockers or clean intermittent catheterization. Three patients subsequently underwent urethral dilation/optical internal urethrotomy for strictures, and 1 patient underwent suprapubic tube placement. Following the failure of these interventions, each of these patients had a UroLume stent placement. A single UroLume stent (2 cm in 3 patients and 2.5 cm in 2 patients) was placed under local/spinal anesthesia. RESULTS: All patients were able to void spontaneously immediately after stent placement. Of the patients with previous urethral strictures, 1 remained continent and 1 had persistent incontinence. Neither of the patients with early postbrachytherapy retention developed incontinence after stent placement. The main complaints following stent placement were referred pain to the head of the penis and dysuria. Stent-related symptoms necessitated stent removal in 2 of 5 patients, 4 to 6 weeks after placement. CONCLUSIONS: The UroLume stent can be used as an alternative form of therapy for managing postbrachytherapy bladder outlet obstruction. The treatment is easily reversible by removing the stent when obstruction resolves.

Hypoxia increases thrombospondin-1 transcript and protein in cultured endothelial cells.

The exposure of endothelial cells to hypoxic environments regulates the expression of a number of genes with products that are vasoactive or mitogenic for vascular tissue, including platelet-derived growth factor, endothelin-1, and endothelial nitric oxide synthase. Hypoxia is also known to alter the adhesive properties of endothelium toward a variety of blood cell types. Thrombospondin-1 (TSP-1) is a glycoprotein with major roles in cellular adhesion and vascular smooth muscle proliferation and migration. We report here that hypoxia induces TSP-1 gene and protein expression. Oxygen tensions of < or =30 torr resulted in TSP-1 transcript induction initially apparent at 1 to 6 hours, with maximal induction (6.5-fold+/-1.2-fold) within 24 to 48 hours in both human and bovine endothelial cells. TSP-1 protein levels remain elevated after 72 hours of continuous hypoxic exposure. The induction of TSP-1 steady-state transcript levels is caused in large part, if not entirely, by post-transcriptional stabilization of the TSP-1 mRNA. The TSP-1 induction by hypoxia is a graded and reversible physiologic response and can be mimicked by the use of cobalt chloride or the inhibition of nitric oxide production, suggesting both the involvement of a heme-containing oxygen sensor and a role for the endogenous production of nitric oxide in TSP-1 regulation. The effects of hypoxia both on the stabilization of the TSP-1 transcript and the stimulation of TSP-1 protein production are completely inhibited by arginine butyrate.

Hypoxia decreases constitutive nitric oxide synthase transcript and protein in cultured endothelial cells.

Endothelial cell-generated nitric oxide (NO) accounts in large part for the labile vasodilator termed endothelium-derived relaxing factor. Two distinct types of NO synthase exist: a "constitutive' type (cNOS), found in endothelial cells, and an "inducible' enzyme. Endothelial cells sense pO2 levels in the range of 70-20 torr and respond to this hypoxia by inducing transcription of genes which encode the vasoactive proteins PDGF-B and endothelin-1. Exposure of human or bovine endothelial cells to low oxygen tensions results in a profound decrease in the transcript for cNOS and a corresponding fall in cNOS protein levels. The ability of endothelial cells exposed to hypoxia to produce NO in response to bradykinin, a stimulator of cNOS activity, was coordinately impaired. Cobalt inhibited the expression of cNOS transcripts, suggesting a mechanism comparable to that by which oxygen tension regulates expression of other vasoregulatory genes. In the presence of actinomycin-D, hypoxia had no effect on cNOS transcripts, suggesting that new gene transcription is required for cNOS suppression. The reducing agents PDTC and N-Ac did not mimic cNOS gene suppression by hypoxia, suggesting that this suppression is not related to the redox state of the intracellular environment. Thus, regulation of cNOS function in response to environmental factors can occur at the level of gene expression as well as at the level of enzyme activation.