Molecular Reactions and Ultrastructural Damage in the Chronically Ischemic Bladder

Molecular Reactions and Ultrastructural Damage in the Chronically Ischemic Bladder Kazem M. Azadzoi,* Bin-Guan Chen, Ziv M. Radisavljevic and Mike B. Siroky From the Departments of Urology (ZMR, MBS) and Pathology, Veterans Affairs Boston Healthcare System (KMA) and Departments of Urology (BGC, MBS) and Pathology, Boston University School of Medicine (KMA), Boston, Massachusetts

Purpose: Clinical and basic research data suggest that pelvic ischemia may contribute to bladder overactivity. We characterized the molecular and ultrastructural reactions of the chronically ischemic bladder. Materials and Method: A model of pelvic ischemia was developed by creating iliohypogastric/pudendal arterial atherosclerosis in rabbits. At 12 weeks conscious urinary frequency was examined, bladder blood flow was recorded and cystometrograms were done using general anesthesia. Bladder tissue was processed for molecular and ultrastructural analysis using quantitative realtime polymerase chain reaction, Western blot and transmission electron microscopy. Results: Conscious urinary frequency and the frequency of spontaneous bladder contractions significantly increased in animals with pelvic ischemia. Bladder ischemia up-regulated the gene and protein expression of hypoxia inducible factor-1␣, transforming growth factor-␤ and nerve growth factor B. Vascular endothelial growth factor gene expression also increased but protein levels were unchanged. Transmission electron microscopy of ischemic bladder samples showed swollen mitochondria with degraded granules, thickened epithelium, deformed muscle fascicles, collagen deposition and impaired microvasculature with thickened intima and disrupted endothelial cell junctions. Degenerating axonal and Schwann cell profiles, and myelin sheath splitting around axons and Schwann cells were evident in ischemic bladders. Conclusions: Interrupting pelvic blood flow resulted in an ischemic overactive bladder and significant increase in conscious urinary frequency. Molecular responses involving hypoxia inducible factor, transforming growth factor-␤, vascular endothelial growth factor and nerve growth factor were associated with mitochondrial injury, fibrosis, microvasculature damage and neurodegeneration. Ischemia may have a key role in bladder overactivity and lower urinary tract symptoms.

Abbreviations and Acronyms HIF ⫽ hypoxia inducible factor LUTS ⫽ lower urinary tract symptoms NGF ⫽ nerve growth factor PCR ⫽ polymerase chain reaction TGF-␤ ⫽ transforming growth factor ␤ VEGF ⫽ vascular endothelial growth factor Submitted for publication January 11, 2011. Study received institutional animal care and use committee approval. Supported by a Department of Veterans Affairs Merit Review Grant. * Correspondence: Surgery/Urology (151), 150 South Huntington Av., Boston, Massachusetts 02130 (telephone: 857-364-5602; FAX: 857-3644540; e-mail: [email protected]).

Key Words: urinary bladder, overactive; ischemia; urination disorders; ultrastructure; gene expression PREVIOUS studies in our model showed that major pelvic artery occlusive disease spread to smaller branches and involved the vesical arteries and bladder arterioles.1,2 This caused decreased blood flow and led to bladder hypoxia and oxidative stress.1,2 A lack of blad-

der perfusion due to arterial insufficiency1,2 and outlet obstruction3 initiated a cascade of hypoxia signaling pathways and led to muscarinic receptor overreactivity and smooth muscle instability. These observations may suggest muscarinic receptor differen-

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Vol. 186, 2115-2122, November 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.06.047

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tial expression and smooth muscle cell hypersensitivity in bladder conditions involving ischemia.1–3 The incidence of vascular insufficiency and ischemic disorders increases with age. Aging associated changes in receptor expression, neural density and neuromuscular interactions are associated with smooth muscle instability in the respiratory, gastrointestinal and cardiovascular systems.4 – 6 This association also appears to hold true for the bladder. The incidence of detrusor instability and LUTS increases with age in men and women.7–9 However, the specific features of aging contributing to LUTS remain unknown. Evidence from clinical and basic research suggests that pelvic vascular disorders and lower urinary tract ischemia may have a role.10 –13 This concept is supported by the clinical correlation of LUTS with peripheral artery atherosclerosis, arteriogenic erectile dysfunction and cardiovascular problems.14,15 There is growing evidence that pelvic ischemia may contribute to bladder overactivity in elderly men and women.10 –18 A significant decrease in bladder blood flow was documented in elderly patients compared with that in asymptomatic younger controls.14,15 Decreased bladder blood flow significantly correlated with LUTS severity in these patients.15 LUTS improvement by ␣-adrenoceptor blockers was associated with significantly increased bladder perfusion.16 Assessment of voiding dysfunction in cardiovascular cases revealed a close correlation between vascular occlusive disorders and the prevalence of LUTS.10,14 Bladder ischemia in humans after over distention and a significant correlation between decreased blood flow and bladder noncompliance were also documented.17,18 In view of these observations we examined bladder contractility, molecular responses and ultrastructural reactions after chronic exposure to ischemia.

MATERIALS AND METHODS Bladder Ischemia Model Animal care and experimental protocols were done in accordance with the guidelines of our institutional animal care and use committee. New Zealand White male rabbits weighing 3 to 3.5 kg were assigned to treatment (10) and age matched control (10) groups. The treatment group was anesthetized with intramuscular ketamine (35 mg/kg) and xylazine (5 mg/kg), followed by isoflurane inhalation (1% to 2% with oxygen). Bladder ischemia was created by the induction of diffused iliohypogastric/pudendal arterial atherosclerosis using a balloon de-endothelialization technique, followed by a short period of a cholesterol diet, as previously described.1,2 At 12 weeks changes in treated animals were compared with those in age matched controls.

Urodynamics and Blood Flow Measurement To examine conscious urinary frequency, under pads were placed in animal cages. Under pad wetting by urination was inspected continuously. When urination was noted, the wet under pads were replaced with dry ones. The number of under pad replacements indicating urinary frequency was recorded for each animal 8 hours daily for 5 days. Average urinary frequency of the treated group was compared with that of age matched controls. Subsequently the animals were anesthetized, as described. Bladder blood flow was recorded with a laser Doppler flowmeter, as we previously reported.1,2 For cystometry a Foley catheter was placed through the urethra into the bladder to infuse normal saline. A 19 gauge angiocatheter was inserted in the bladder for intravesical pressure measurement. The bladder was filled continuously with saline at a rate of 0.8 ml per minute. The volume that induced micturition was recorded and intravesical pressure at micturition was measured. The Foley catheter balloon was then inflated to prevent leakage and the bladder was filled with 30 ml normal saline. The frequency of spontaneous bladder contraction was measured as the number of contractions per 10 minutes. The bladders were then removed and tissue samples were analyzed, as described.

Quantitative Real-Time PCR We examined the gene expression of HIF-1␣, TGF-␤, VEGF and NGFB. Sequences of HIF-1␣ (GenBank® AY273790), TGF-␤ (GenBank AF000133), VEGF (GenBank AY196796) and NGFB (GenBank NM002506) were used to design TaqMan® probes. HIF-1␣, TFG-␤, VEGF and NGFB gene sequences were tested for repetitive sequences and masked when detected. RNA was isolated from bladder tissues and 50 ng RNA were processed for 2-step quantitative PCR using an ABI Prism® 7700. TaqMan amplicon probes with forward and reverse primers were developed and 18s served as the internal control. All samples were amplified in duplicate. Gene expression in treated bladder samples was calculated as the relative quantification to age matched control values in folds.

Western Blot Bladder tissues were homogenized and centrifuged, and pellets were discarded. Equal amounts of protein lysate (100 ␮g) were processed for 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis for 1 hour 45 minutes. Gels were blotted on immunoblot polyvinylidene fluoride (Bio-Rad®) for 4 hours. Membranes were blocked by 10% nonfat milk, incubated with primary antibodies against HIF, TFG-␤, VEGF and NGF or ␣-actin (Santa Cruz Biotechnology, Santa Cruz, California) for 2 hours and washed 3 times with tris buffered saline. Membranes were incubated with fluorescent secondary antibody, scanned and analyzed by Typhoon™ image analysis software. To track the molecular weight of individual proteins Precision Plus Protein™ All Blue Standards were included in each sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel.

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No. conscious micturitions/8 hrs Bladder blood flow (ml/min/100 gm) Bladder at micturition: Vol (ml) Pressure (mm Hg) No. contractions/10 mins Pressure increase at contraction (mm Hg)

Mean ⫾ SEM Control

Mean ⫾ SEM Treatment

2.4 ⫾ 0.3 6.6 ⫾ 0.8

4.6 ⫾ 0.5 2.9 ⫾ 0.5

23 ⫾ 4.2 15 ⫾ 2.0 1.2 ⫾ 0.4 4.1 ⫾ 1.5

17 ⫾ 3.8 18 ⫾ 2.7 6.1 ⫾ 1.2 5.9 ⫾ 1.8

Transmission Electron Microscopy Bladder tissues were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, washed in 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide. Samples were washed, incubated in 1% aqueous uranyl acetate, dehydrated in grades of alcohol, placed in propylene oxide for 1 hour and infiltrated in a 1:1 mixture of propylene oxide. The next day the samples were embedded and polymerized. Ultrathin sections were cut and picked up on copper grids stained with lead citrate. The ultrastructural quality of ischemic cellular and subcellular components was compared with that of control samples using a JEOL® 1200EX microscope.

Statistical Analysis Data are shown as the mean ⫾ SEM. Significant differences between the treated and control groups were determined by ANOVA, followed by post hoc comparisons. Statistical significance was considered at p ⱕ0.05.

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RESULTS Bladder Ischemia Urodynamic Changes Arterial atherosclerosis in the treated group produced significant bladder ischemia (see table). Conscious urinary frequency in animals with bladder ischemia was significantly greater than that in age matched controls (see table). In anesthetized animals bladder volume at micturition tended to be less and intravesical pressure tended to be higher in ischemic bladders but these values did not attain significance due to large SDs (see table). The frequency of spontaneous bladder contractions was significantly greater in ischemic than in control bladders. The intravesical pressure increase during contraction tended to be higher in ischemic bladders but it varied largely among the animals and did not attain significance compared with that in controls (see table). Molecular Reactions to Ischemia Bladder ischemia initiated molecular signaling, indicative of hypoxia, fibrosis, and degeneration of the microvasculature and nerve fibers. Quantitative real time-PCR revealed significant up-regulation of HIF-1␣, TGF-␤, VEGF and NGFB in ischemic bladder tissue comparison to that in age matched controls (figs. 1 and 2).

Figure 1. Real-time PCR and Western blot revealed significant increases in HIF and TGF-␤ gene and protein expression in ischemic bladder tissue. Data are shown as mean ⫾ SEM. Asterisk indicates significantly different vs controls. OD, optical density.

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Figure 2. Real-time PCR showed significant increases in VEGF and NGF gene expression in ischemic bladder tissue. Western blot demonstrated increased NGF protein expression in ischemic bladder but unchanged VEGF protein. Data are shown as mean ⫾ SEM. Asterisk indicates significantly different vs controls.

Protein Expression Changes The protein expression of HIF, TGF-␤, VEGF and NGF did not always follow the gene expression pattern described. While HIF-1␣, TGF-␤ and NGF protein significantly increased in ischemic bladders, protein expression was unchanged compared with that in age matched controls (figs. 1 and 2). Ultrastructural Reactions Transmission electron microscopy of ischemic tissues revealed thickened epithelium, deformed muscle fascicles and increased collagen deposition (fig. 3). Subcellular changes included swollen mitochondria with degraded granules, partial loss of mitochondrial membrane and sporadic vacuolization. The ischemic bladder microvasculature showed a thickened intima, diffused subintimal fibrosis, disruption of vascular endothelial cell junctions and sporadic loss of arteriolar endothelial cells (fig. 4). Degenerating axons with collapsed axon and Schwann cell profiles surrounded by dense connective tissue and splitting of the myelin sheath around axon and Schwann cells were evident in ischemic bladder tissue (fig. 4).

DISCUSSION In previous studies we found that occlusive disease of the major pelvic arteries spread to smaller

branches and disrupted the perfusion of pelvic organs, including the bladder.1,2 Our current study shows that interrupting pelvic blood flow increased conscious urinary frequency and led to an overactive bladder with distinctive molecular and ultrastructural reactions. Bladder overactivity in our model was assessed based on changes in intravesical pressure and the increased frequency of spontaneous bladder contractions. A limitation of our current study relates to the lack of bladder outflow data to enable urethral resistance assessment. Ischemia could affect bladder contractility directly and indirectly by increasing urethral resistance. In our previous studies in the model decreased pelvic blood flow resulted in significant bladder hypoxia, free radical activity and the accumulation of oxidative products.1,2 These ischemic and hypoxic changes were associated with decreased bladder neural density, altered muscarinic receptor reactivity and increased smooth muscle contractions in response to muscarinic receptor agonists and electrical field stimulation.1,2 The mechanism appeared to involve oxidative stress and it increased the production of cyclooxygenase and lipoxygenase products.1,2 In the current study chronic ischemia initiated molecular reactions involving HIF, TGF-␤, VEGF and NGF, and led to widespread structural damage

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Figure 3. Transmission electron microscopy shows epithelial and smooth muscle layers in tissue samples of ischemic and age matched control bladders. White arrows indicate mitochondria with distinct membrane and granules in control, and mitochondria with swollen membrane and disrupted granules in ischemic tissue. Black arrows indicate thickened folded mucosal membrane in ischemic tissue vs control. Note sporadic vacuolization in ischemic epithelium. Two-headed arrows indicate dense smooth muscle bundles with well defined nucleus and normal connective tissue distribution in control, and deformed smooth muscle fascicles with increased collagen deposition in ischemic tissue. Reduced from ⫻23,000 (epithelium) and ⫻19,000 (smooth muscle).

in bladder mucosa, smooth muscle cells, microvasculature and nerve fibers. Molecular reactions to ischemia were more distinctive at the transcriptional stage compared with changes at the transitional level. While increased gene expression of HIF, TGF-␤ and NGF correlated with their protein expression, up-regulation of the VEGF gene apparently failed to increase its protein levels under ischemic conditions. These molecular events could activate a cascade of downstream pathways and lead to contractile dysfunction and progressive degeneration of the bladder wall. Observations in our model in conjunction with the clinical correlation of bladder ischemia with LUTS may suggest a role for pelvic arterial insufficiency in detrusor overactivity and voiding dysfunction. Factors contributing to ischemic bladder damage and dysfunction may include nutrient deficiency, hypoxia, lack of perfusion to remove metabolic waste, and the accumulation of cytotoxic and neurotoxic elements. Metabolic waste is normally cleared by tissue perfusion at systemic arterial pressure. Free radicals are tightly regulated by homeostatic mechanisms. In the healthy bladder the cellular an-

tioxidant defense system in conjunction with dietary antioxidants neutralize free radicals to protect tissues from oxidative injury. In bladder ischemia nutrient deficiency, hypoxia and the lack of perfusion result in the accumulation of waste products and provide an environment that is hospitable to free radicals and oxidative reactions. This appears to initiate a cascade of molecular events with a profound impact on the structural integrity of cell organelles, smooth muscle cells, microvasculature and nerve fibers. Similar changes were documented in experimental bladder outlet obstruction models.3,19,20 Increasing bladder pressure with filling caused significantly greater ischemia and hypoxia in obstructed bladders than in controls.3 Intermittent hypoxia resulted in significant hypoxyprobe-1–protein adducts in the detrusor of the obstructed bladder compared with those in age matched controls.19,20 Partial bladder outlet obstruction initiated selective metabolic dysfunction of the smooth muscle cells, which was characterized by decreases in mitochondrial function and sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase activity.20

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Figure 4. Transmission electron microscopy demonstrates microvasculature and nerve fibers in tissue samples of ischemic and age matched control bladders. Black arrows indicate normal intima with well-defined endothelial cell lining in control and swollen intima with disrupted endothelial cells in ischemic tissue. White arrows indicate normal endothelial cell junction in control and disrupted endothelial cell junction with peculiar folding in ischemic sample. Black 2-headed arrows indicate marked thickening and fibrosis of subintimal layer in ischemic sample vs control. White 2-headed arrows indicate nerve fiber with normal profile in control and degenerating nerve fiber surrounded by collagen in ischemic sample. Reduced from ⫻18,500 (microvasculature) and ⫻4,800 (nerve fibers).

Our data in conjunction with observations in the obstructed model suggest that the bladder is highly reactive to conditions that influence its blood perfusion. Intermittent ischemia initiates noxious free radical activity mediated by O2⫺, which rapidly interacts with nitric oxide to generate additional radicals and instigate nitrosative activity.21 The lack of perfusion and the subsequent accumulation of oxidative and nitrosative elements in the ischemic bladder1,2 may contribute to the deterioration of epithelium, smooth muscle cells, microvasculature and nerve fibers by lipid peroxidation, protein oxidation and DNA damage.19 –21 Up-regulation of HIF-1␣ gene and protein expression in the ischemic bladder may suggest a tissue response to low oxygen tension and could initiate ischemic cell survival signaling to stipulate downstream defensive reactions against hypoxia and free radical incursion. Our data are consistent with the reported up-regulation of HIF-1␣ in association with an inflammatory response at the initial stages of bladder outlet obstruction.22 This response in the obstructed bladder is thought to be initiated by intermittent hypoxia and it is followed by smooth mus-

cle hypertrophy and fibrosis.22 Increased HIF gene and protein expression in our model was associated with specific changes in the mitochondrial membrane and granules reported in oxidative stress conditions.21 Mitochondrial damage may contribute to the formation of additional free radicals and lead to oxidative incursion of surrounding cellular components. Oxidative reactions in mitochondrial complexes I to V of energy production and the subsequent disruption of mitochondrial granules may impair other cell organelles and contribute to smooth muscle cell degeneration in the ischemic bladder. Under ischemic conditions when oxygen tension is low, some electrons passing through the mitochondrial electron transport chain react with molecular oxygen. This results in the O2⫺ anion, which rapidly interacts with surrounding molecules and generates additional free radicals through enzymatic and nonenzymatic mechanisms.23,24 Aging associated degenerative disorders involve the mitochondrial release of degenerating factors, including cytochrome c, endonuclease G and apoptosis inducing factor to the cytosol.24,25 These regulators activate a cascade of cell survival signaling

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pathways, leading to cell organelle deterioration and the progressive degeneration of cellular structures. Excessive formation of free radicals and increased oxidative activity in the ischemic bladder could activate similar signaling pathways and contribute to degenerative changes in epithelium, smooth muscle, microvasculature and nerve fibers (figs. 3 and 4). Up-regulation of TGF-␤ gene and protein expression was accompanied by structural changes in smooth muscle cells, increased connective tissue, collagen invasion of nerve fibers and sporadic vacuolization of the ischemic bladder layers. TGF-␤ is implicated in the induction of collagen deposition and soft tissue fibrosis at the decompensation phase of the partially obstructed bladder.22 TGF-␤ regulates extracellular matrix synthesis and degradation, and balances the relative amount of smooth muscle and connective tissue.26 The ischemia associated collagen deposition documented in our model impairs the fibroelastic properties of the bladder wall and contributes to noncompliance.27 These observations are consistent with a clinical report of a significant correlation between decreased human bladder blood flow and noncompliance.18 Our data are supported by reports of ischemia associated TGF-␤ up-regulation in aortic smooth muscle cells.26 It is thought that under hypoxic conditions TGF-␤ up-regulates its own mRNA and increases its receptor expression.26 While VEGF gene expression significantly increased in response to bladder ischemia, its protein levels remained unchanged. Up-regulation of VEGF gene expression may be a defensive reaction to stimulate neoangiogenesis or repair microvasculature endothelial damage, subintimal fibrosis, the disruption of endothelial cell junctions and the loss of vascular endothelial cells in the ischemic bladder. Similar changes in VEGF expression were reported 2 weeks after the induction of bladder outlet obstruction.28 The vascular growth factor reaction in the obstructed bladder is thought to have a role in cellular remodeling.28 However, in our model the up-regulation of VEGF gene expression did not increase its protein levels or achieve neoangiogenesis and it apparently failed to protect the bladder microvasculature against ischemic injury. A possibility is that smooth muscle and endothelial cells may not be capable of synthesizing VEGF protein in ischemic tissues under chronic hypoxia and oxidative stress conditions. Other possibilities may relate to the containment of VEGF synthesis by TGF-␤ and the induction of collagen synthesis. Studies of vascular tissues show that ischemia and hypoxia are potent stimulators of angiogenesis via angiogenic growth factors and proteins that induce endothelial and smooth muscle cell proliferation and migration.29 VEGF serves as a sur-

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vival factor for endothelial cells with cell specific mitogen properties in vitro and angiogenic activity in vivo.29 Experimental inhibition of VEGF resulted in deterioration and detachment of endothelial cells from blood vessels.29 Vascular studies suggest that HIF up-regulation under hypoxic conditions may regulate the induction of VEGF transcription to stimulate neoangiogenesis.29 NGF gene and protein expression were also significantly up-regulated in the ischemic bladder. Transmission electron microscopy of ischemic bladder samples revealed a loss of neural structural integrity with degenerating changes in the axonal profile, Schwann cells and the myelin sheath. NGF up-regulation in the ischemic bladder may also be an intrinsic defensive response to protect neural structures against hypoxia and free radical injury. However, this maneuver appeared to fail to protect neural structural integrity under ischemic bladder conditions. In a previous study we found that gene expression of p75 NGF receptor was down-regulated at the early stages of bladder ischemia immediately after the induction of arterial insufficiency.1 Rapid reaction and impairment of NGF receptors may explain the failure of NGF gene expression to stimulate new neural outgrowth and prevent neurodegeneration in the ischemic bladder model. A lack of perfusion may affect NGF expression and neural structures directly by nutrient deficiency and hypoxia or indirectly by the accumulation of free radicals and oxidative products in nerve fibers and surrounding tissues. The role of ischemia and oxidative stress in the pathogenesis of aging related neurodegenerative disorders has been reported.30 NGF prevents or reverses the sensory deficit, myelin degeneration and dysfunctional transmitter release in nonischemic tissues.30 NGF action on impaired nerves involves the induction of angiogenic factors in Schwann cells and neurons.30 These studies suggest close intercommunication among HIF, VEGF and NGF to enhance neoangiogenesis to provide nutrients for the protection of impaired nerves and the stimulation of new neural outgrowth.29,30 Oxidative stress and peroxynitrite inhibited the NGF prosurvival signal and led to neurodegeneration in diabetic models.30

CONCLUSIONS Interruption of the arterial supply to the pelvis led to an ischemic overactive bladder with repeating cycles of spontaneous contractions and caused a significant increase in conscious urinary frequency. HIF, TGF-␤, VEGF and NGF up-regulation in the ischemic bladder was accompanied by the loss of mitochondrial structural integrity, fibrosis, and the

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degeneration of microvasculature and nerve fibers. These observations may suggest the role of ischemia in the overactive bladder with impaired contraction,

as reported in elderly patients without obstruction. Ischemia may be a key factor in aging associated LUTS.

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