Cotadutide improves brown adipose tissue thermogenesis in obese mice.

We studied the effect of cotadutide, a dual agonist glucagon-like peptide 1 (GLP1)/Glucagon, on interscapular brown adipose tissue (iBAT) remodeling and thermogenesis of obese mice. Twelve-week-old male C57BL/6 mice were fed a control diet (C group, n = 20) or a high-fat diet (HF group, n = 20) for ten weeks. Then, animals were redivided, adding cotadutide treatment: C, CC, HF, and HFC for four additional weeks. The multilocular brown adipocyte structure showed fat conversion (whitening), hypertrophy, and structural disarray in the HF group, which was reverted in cotadutide-treated animals. Cotadutide enhances the body temperature, thermogenesis, and sympathetic innervation (peroxisome proliferator-activated receptor-α, ß3 adrenergic receptor, interleukin 6, and uncoupled protein 1), reduces pro-inflammatory markers (disintegrin and metallopeptidase domain, morphogenetic protein 8a, and neuregulin 4), and improves angiogenesis (vascular endothelial growth factor A, and perlecan). In addition, cotadutide enhances lipolysis (perilipin and cell death-inducing DNA fragmentation factor α), mitochondrial biogenesis (nuclear respiratory factor 1, transcription factor A mitochondrial, mitochondrial dynamin-like GTPase, and peroxisome proliferator-activated receptor gamma coactivator 1α), and mitochondrial fusion/fission (dynamin-related protein 1, mitochondrial fission protein 1, and parkin RBR E3 ubiquitin protein ligase). Cotadutide reduces endoplasmic reticulum stress (activating transcription factor 4, C/EBP homologous protein, and growth arrest and DNA-damage inducible), and extracellular matrix markers (lysyl oxidase, collagen type I α1, collagen type VI α3, matrix metallopeptidases 2 and 9, and hyaluronan synthases 1 and 2). In conclusion, the experimental evidence is compelling in demonstrating cotadutide's thermogenic effect on obese mice's iBAT, contributing to unraveling its action mechanisms and the possible translational benefits.

Treatment with semaglutide, a GLP-1 receptor agonist, improves extracellular matrix remodeling in the pancreatic islet of diet-induced obese mice.

AIMS: The extracellular matrix (ECM) is fundamental for the normal endocrine functions of pancreatic islet cells and plays key roles in the pathophysiology of type 2 diabetes. Here we investigated the turnover of islet ECM components, including islet amyloid polypeptide (IAPP), in an obese mouse model treated with semaglutide, a glucagon-like peptide type 1 receptor agonist. MAIN METHODS: Male one-month-old C57BL/6 mice were fed a control diet (C) or a high-fat diet (HF) for 16 weeks, then treated with semaglutide (subcutaneous 40 µg/kg every three days) for an additional four weeks (HFS). The islets were immunostained and gene expressions were assessed. KEY FINDINGS: Comparisons refer to HFS vs HF. Thus, IAPP immunolabeling and beta-cell-enriched beta-amyloid precursor protein cleaving enzyme (Bace2, -40 %) and heparanase immunolabeling and gene (Hpse, -40 %) were mitigated by semaglutide. In contrast, perlecan (Hspg2, +900 %) and vascular endothelial growth factor A (Vegfa, +420 %) were enhanced by semaglutide. Also, semaglutide lessened syndecan 4 (Sdc4, -65 %) and hyaluronan synthases (Has1, -45 %; Has2, -65 %) as well as chondroitin sulfate immunolabeling, and collagen type 1 (Col1a1, -60 %) and type 6 (Col6a3, -15 %), lysyl oxidase (Lox, -30 %) and metalloproteinases (Mmp2, -45 %; Mmp9, -60 %). SIGNIFICANCE: Semaglutide improved the turnover of islet heparan sulfate proteoglycans, hyaluronan, chondroitin sulfate proteoglycans, and collagens in the islet ECM. Such changes should contribute to restoring a healthy islet functional milieu and should reduce the formation of cell-damaging amyloid deposits. Our findings also provide additional evidence for the involvement of islet proteoglycans in the pathophysiology of type 2 diabetes.

Semaglutide (GLP-1 receptor agonist) stimulates browning on subcutaneous fat adipocytes and mitigates inflammation and endoplasmic reticulum stress in visceral fat adipocytes of obese mice.

Semaglutide (GLP-1 agonist) was approved for treating obesity. Although the effects on weight loss and metabolism are known, the responses of adipocytes to semaglutide are yet limited. C57BL/6 male mice (n = 20/group) were fed a control diet (C) or a high-fat (HF) diet for 16 weeks and then separated into four groups (n = 10/group) for an additional four weeks: C, C diet and semaglutide, HF, and HF diet and semaglutide. Epididymal white adipose tissue (eWAT) and subcutaneous white adipose tissue (sWAT) fat pads were studied with biochemistry, immunohistochemistry/fluorescence, stereology, and reverse transcription-quantitative polymerase chain reaction. In obese mice, semaglutide reduced the fat pad masses (eWAT, -55%; sWAT, -40%), plasmatic cytokines, and proinflammatory gene expressions: tumor necrosis factor-alpha (-60%); interleukin (IL)-6 (-55%); IL-1 beta (-40%); monocyte chemoattractant protein-1 (-90%); and leptin (-80%). Semaglutide also lessened endoplasmic reticulum (ER) stress genes of activating transcription factor-4 (-85%), CCAAT enhancer-binding protein homologous protein (-55%), and growth arrest and DNA damage-inducible gene 45 (-45%). The obese mice's adipocyte hypertrophy and macrophage infiltration were equally reduced by semaglutide. Semaglutide enhanced multiloculation and uncoupled protein 1 (UCP1) labeling in obese mice: peroxisome proliferator-activated receptor-alpha (+560%) and gamma (+150%), fibronectin type III domain-containing protein 5 (+215%), peroxisome proliferator-activated receptor-alpha coactivator (+110%), nuclear respiratory factor 1 (+260%), and mitochondrial transcription factor A (+120%). Semaglutide also increased thermogenetic gene expressions for the browning phenotype maintenance: beta-3 adrenergic receptor (+520%), PR domain containing 16 (+90%), and Ucp1 (+110%). In conclusion, semaglutide showed significant beneficial effects beyond weight loss, directly on fat pads and adipocytes of obese mice, remarkably anti-inflammatory, and reduced adipocyte size and ER stress. Besides, semaglutide activated adipocyte browning, improving UCP1, mitochondrial biogenesis, and thermogenic marker expressions help weight loss.

Study of the ureter structure in anencephalic fetuses

Purpose The objective of this paper is to analyze the structure of the ureter in normal and anencephalic human fetuses. Materials and Methods We studied 16 ureters from 8 human fetuses without congenital anomalies aged 16 to 27 weeks post-conception (WPC) and 14 ureters from 7 anencephalic fetuses aged 19 to 33 WPC. The ureters were dissected and embedded in paraffin, from which 5 µm thick sections were obtained and stained with Masson trichrome, to quantify smooth muscle cells (SMC) and to determine the ureteral lumen area, thickness and ureteral diameter. The samples were also stained with Weigert Resorcin Fucsin (to study elastic fibers) and Picro-Sirius Red with polarization and immunohistochemistry analysis of the collagen type III fibers to study collagen. Stereological analysis of collagen, elastic system fibers and SMC were performed on the sections. Data were expressed as volumetric density (Vv-%). The images were captured with an Olympus BX51 microscope and Olympus DP70 camera. The stereological analysis was done using the Image Pro and Image J programs. For biochemical analysis, samples were fixed in acetone, and collagen concentrations were expressed as micrograms of hydroxyproline per mg of dry tissue. Means were statistically compared using the unpaired t-test (p < 0.05). Results The ureteral epithelium was well preserved in the anencephalic and control groups. We did not observe differences in the transitional epithelium in the anencephalic and control groups. There was no difference in elastic fibers and total collagen distribution in normal and anencephalic fetuses. SMC concentration did not differ significantly (p = 0.1215) in the anencephalic and control group. The ureteral lumen area (p = 0.0047), diameter (p = 0.0024) and thickness (p = 0.0144) were significantly smaller in anencephalic fetuses. Conclusions Fetuses with anencephaly showed smaller diameter, area and thickness. These differences could indicate ...

Protective effects of l-glutamine on the bladder wall of rats submitted to pelvic radiation.

Radiotherapy is often used to treat prostate tumors, but the normal bladder is usually adversely affected. Using an animal model of pelvic radiation, we investigated whether glutamine nutritional supplementation can prevent radiation-induced damage to the bladder, especially in its more superficial layers. Male rats aged 3-4 months were divided into groups of 8 animals each: controls, which consisted intact animals; radiated-only rats, which were sacrificed 7 (R7) or 15 (R15) days after a radiation session (10Gy aimed at the pelvico-abdominal region); and radiated rats receiving l-glutamine supplementation (0.65g/kg body weight/day), which were sacrificed 7 (RG7) or 15 (RG15) days after the radiation session. Cells and blood vessels in the vesical lamina propria, as well as the urothelium, were then measured using histological methods. The effects of radiation were evaluated by comparing controls vs. either R7 or R15, while a protective effect of glutamine was assessed by comparing R7 vs. RG7 and R15 vs. RG15. The results showed that, in R7, epithelial thickness, epithelial cell density, and cell density in the lamina propria were not significantly affected. However, density of blood vessels in R7 was reduced by 48% (p<0.05) and this alteration was mostly prevented by glutamine (p<0.02). In R15, density of blood vessels in the lamina propria was not significantly modified. However, epithelial thickness was reduced by 25% (p<0.05) in R15, and this effect was prevented by glutamine (p<0.01). In R15, epithelial cell density was increased by 35% (p<0.02), but glutamine did not protect against this radiation-induced increase. Cell density in the lamina propria was likewise unaffected in R15. Density of mast cells in the lamina propria was markedly reduced in R7 and R15. The density was still reduced in RG7, but a higher density in RG15 suggested a glutamine-mediated recovery. Alpha-actin positive cells in the lamina propria formed a suburothelial layer and were identified as myofibroblasts. Thickness of this layer was increased in R7, but was similar to controls in RG7, while changes in R15 and RG15 were less evident. In conclusion, pelvic radiation leads to significant acute and post-acute alterations in the composition and structural features of the vesical lamina propria and epithelium. Most of these changes, however, can be prevented by glutamine nutritional supplementation. These results emphasize, therefore, the potential use of this aminoacid as a radioprotective drug.

Does prolonged pneumoperitoneum affect the kidney? Oxidative stress, stereological and electron microscopy study in a rat model.

PURPOSE: Pneumoperitoneum (Pp) at 12 to 15 mmHg in rats is associated with kidney damage. However, Pp at 8 mmHg is now known to best correlate to working pressures used in humans. Thus the aim of this work was to study the kidney of rats submitted to prolonged Pp at 8 mmHg. MATERIALS AND METHODS: Rats were divided into a Sham group (n = 14), submitted to anesthesia, and a Pp group (n = 14), submitted to Pp at 8 mmHg, followed by deflation. In both groups, 7 animals were immediately killed and their kidneys were used for oxidative stress analyses. The remaining 7 rats in each group were evaluated after 6 weeks for the number of glomeruli and podocyte morphology. RESULTS: For all analyzed parameters Sham and Pp groups presented no statistical difference. CONCLUSION: When submitted to adequate Pp pressures (8 mmHg), no kidney damage occurs in rats.

Does prolonged pneumoperitoneum affect the kidney? Oxidative stress, stereological and electron microscopy study in a rat model

Purpose: Pneumoperitoneum (Pp) at 12 to 15 mmHg in rats is associated with kidney damage. However, Pp at 8 mmHg is now known to best correlate to working pressures used in humans. Thus the aim of this work was to study the kidney of rats submitted to prolonged Pp at 8 mmHg. Materials and Methods: Rats were divided into a Sham group (n = 14), submitted to anesthesia, and a Pp group (n = 14), submitted to Pp at 8 mmHg, followed by deflation. In both groups, 7 animals were immediately killed and their kidneys were used for oxidative stress analyses. The remaining 7 rats in each group were evaluated after 6 weeks for the number of glomeruli and podocyte morphology. Results: For all analyzed parameters Sham and Pp groups presented no statistical difference. Conclusion: When submitted to adequate Pp pressures (8 mmHg), no kidney damage occurs in rats. .

Nutritional supplementation with L-arginine prevents pelvic radiation-induced changes in morphology, density, and regulating factors of blood vessels in the wall of rat bladder.

PURPOSE: To determine whether L-arginine has protective effects against radiation-induced alterations in the morphology and regulatory factors of vesical blood vessels in rats. METHODS: Male rats aged 3-4 months were divided into groups of 10 animals each: (a) controls, consisting of non-treated animals; (b) radiated-only rats; and (c) radiated rats receiving L-arginine supplementation. Radiation was in one session of 10 Gy and was aimed at the pelvic-abdominal region. L-arginine was administered once a day (0.65 g/kg body weight), starting 7 days before radiation and continuing until killing on the 16th day after radiation. The density, relative area, and wall thickness of blood vessels were measured in the vesical lamina propria using histological methods, and the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGF) in the bladder wall was assessed by RT-PCR. RESULTS: Compared with controls, radiation alone decreased the density and relative area of blood vessels by 32 % (p < 0.01) and 25 % (p < 0.05), respectively, and reduced the arterial wall thickness by 42 % (p < 0.004). VEGF and FGF mRNA levels after radiation were diminished by 67 % (p < 0.002) and 56 % (p < 0.04), respectively. The radiated animals supplemented with L-arginine were not significantly different from controls. CONCLUSIONS: Pelvic radiation leads to significant vesical modifications, as in the morphology of blood vessels and in VEGF and FGF expression. All these changes, however, were prevented by L-arginine treatment. These results emphasize, therefore, the potential use of this amino acid as a radioprotective drug.

Study of the ureter structure in anencephalic fetuses.

PURPOSE: The objective of this paper is to analyze the structure of the ureter in normal and anencephalic human fetuses. MATERIALS AND METHODS: We studied 16 ureters from 8 human fetuses without congenital anomalies aged 16 to 27 weeks post-conception (WPC) and 14 ureters from 7 anencephalic fetuses aged 19 to 33 WPC. The ureters were dissected and embedded in paraffin, from which 5 µm thick sections were obtained and stained with Masson trichrome, to quantify smooth muscle cells (SMC) and to determine the ureteral lumen area, thickness and ureteral diameter. The samples were also stained with Weigert Resorcin Fucsin (to study elastic fibers) and Picro-Sirius Red with polarization and immunohistochemistry analysis of the collagen type III fibers to study collagen. Stereological analysis of collagen, elastic system fibers and SMC were performed on the sections. Data were expressed as volumetric density (Vv-%). The images were captured with an Olympus BX51 microscope and Olympus DP70 camera. The stereological analysis was done using the Image Pro and Image J programs. For biochemical analysis, samples were fixed in acetone, and collagen concentrations were expressed as micrograms of hydroxyproline per mg of dry tissue. Means were statistically compared using the unpaired t-test (p < 0.05). RESULTS: The ureteral epithelium was well preserved in the anencephalic and control groups. We did not observe differences in the transitional epithelium in the anencephalic and control groups. There was no difference in elastic fibers and total collagen distribution in normal and anencephalic fetuses. SMC concentration did not differ significantly (p = 0.1215) in the anencephalic and control group. The ureteral lumen area (p = 0.0047), diameter (p = 0.0024) and thickness (p = 0.0144) were significantly smaller in anencephalic fetuses. CONCLUSIONS: Fetuses with anencephaly showed smaller diameter, area and thickness. These differences could indicate that anencephalic fetal ureters tend to have significant structural alterations, probably due to cerebral lesions with consequent brain control damage of ureter nerves.

Stereological and biochemical analysis of the urethral edges in patients submitted to end-to-end anastomosis for bulbar urethral stricture.

PURPOSE: To study the morphologic alterations in the proximal and distal urethral edges from patients submitted to end-to-end bulbar urethroplasty. MATERIALS AND METHODS: We analyzed 12 patients submitted to anastomotic urethroplasty to treat bulbar strictures less than 2.0 cm in length. After excision of the fibrotic segment to a 28Fr urethral caliber, we obtained biopsies from the spongious tissue of the free edges (proximal: PROX and distal: DIST). Controls included normal bulbar urethras obtained from autopsies of 10 age matched individuals. The samples were histologically processed for smooth muscle cells (SMC), elastic system fibers and collagen. Stereological analysis was performed to determine the volumetric density (Vv) of each element. Also, a biochemical analysis was performed to quantify the total collagen content. RESULTS: Vv of SMC was reduced in PROX (31.48 ± 7.01 p < 0.05) and similar in DIST when compared to controls (55.65 ± 9.60%) with no statistical difference. Elastic fibers were increased in PROX (25.70 ± 3.21%; p < 0.05) and were similar to controls in DIST (15.87 ± 4.26%). Total collagen concentration in PROX (46.39 ± 8.20 µg/mg), and DIST (47.96 ± 9.42 µg/mg) did not differ from controls (48.85 ± 6.91 µg/mg). Type III collagen was similarly present in all samples. CONCLUSIONS: After excision of the stenotic segment to a caliber of 28Fr, the exposed and macroscopically normal urethral edges may present altered amounts of elastic fibers and SMC, but are free from fibrotic tissue. When excising the peri-stenotic tissue, the surgeon should be more careful in the proximal end, which is the most altered.

Stereological and biochemical analysis of the urethral edges in patients submitted to end-to-end anastomosis for bulbar urethral stricture

PURPOSE: To study the morphologic alterations in the proximal and distal urethral edges from patients submitted to end-to-end bulbar urethroplasty. MATERIALS AND METHODS: We analyzed 12 patients submitted to anastomotic urethroplasty to treat bulbar strictures less than 2.0 cm in length. After excision of the fibrotic segment to a 28Fr urethral caliber, we obtained biopsies from the spongious tissue of the free edges (proximal: PROX and distal: DIST). Controls included normal bulbar urethras obtained from autopsies of 10 age matched individuals. The samples were histologically processed for smooth muscle cells (SMC), elastic system fibers and collagen. Stereological analysis was performed to determine the volumetric density (Vv) of each element. Also, a biochemical analysis was performed to quantify the total collagen content. RESULTS: Vv of SMC was reduced in PROX (31.48 ± 7.01 p < 0.05) and similar in DIST when compared to controls (55.65 ± 9.60%) with no statistical difference. Elastic fibers were increased in PROX (25.70 ± 3.21%; p < 0.05) and were similar to controls in DIST (15.87 ± 4.26%). Total collagen concentration in PROX (46.39 ± 8.20 μg/mg), and DIST (47.96 ± 9.42 μg/mg) did not differ from controls (48.85 ± 6.91 μg/mg). Type III collagen was similarly present in all samples. CONCLUSIONS: After excision of the stenotic segment to a caliber of 28Fr, the exposed and macroscopically normal urethral edges may present altered amounts of elastic fibers and SMC, but are free from fibrotic tissue. When excising the peri-stenotic tissue, the surgeon should be more careful in the proximal end, which is the most altered.

Structural alterations of foreskin caused by chronic smoking may explain high levels of urethral reconstruction failure using foreskin flaps.

OBJECTIVES: The aim of the present study was to perform a stereological and biochemical analysis of the foreskin of smoker subjects. MATERIALS AND METHODS: Foreskin samples were obtained from 20 young adults (mean = 27.2 years old) submitted to circumcision. Of the patients analyzed, one group (n = 10) had previous history of chronic smoking (a half pack to 3 packs per day for 3 to 13 years (mean = 5.8 ± 3.2). The control group included 10 nonsmoking patients. Masson 's trichrome stain was used to quantify the foreskin vascular density. Weigert's resorcin-fucsin stain was used to assess the elastic system fibers and Picrosirius red stain was applied to study the collagen. Stereological analysis was performed using the Image J software to determine the volumetric densities. For biochemical analysis, the total collagen was determined as µg of hydroxyproline per mg of dry tissue. Means were compared using the unpaired t-test (p < 0.05). RESULTS: Elastic system fibers of smokers was 42.5 % higher than in the control group (p = 0.002). In contrast, smooth muscle fibers (p = 0.42) and vascular density (p = 0.16) did not show any significant variation. Qualitative analysis using Picrosirius red stain with polarized light evidenced the presence of type I and III collagen in the foreskin tissue, without significant difference between the groups. Total collagen concentration also did not differ significantly between smokers and non-smokers (73.1 µg/mg ± 8.0 vs. 69.2 µg/mg ± 5.9, respectively, p = 0.23). CONCLUSIONS: The foreskin tissue of smoking patients had a significant increase of elastic system fibers. Elastic fibers play an important role in this tissue's turnover and this high concentration in smokers possibly causes high extensibility of the foreskin. The structural alterations in smokers' foreskins could possibly explain the poor results in smoking patients submitted to foreskin fasciocutaneous flaps in urethral reconstruction surgery.

Structural alterations of foreskin caused by chronic smoking may explain high levels of urethral reconstruction failure using foreskin flaps

OBJECTIVES: The aim of the present study was to perform a stereological and biochemical analysis of the foreskin of smoker subjects. MATERIALS AND METHODS: Foreskin samples were obtained from 20 young adults (mean = 27.2 years old) submitted to circumcision. Of the patients analyzed, one group (n = 10) had previous history of chronic smoking (a half pack to 3 packs per day for 3 to 13 years (mean = 5.8 ± 3.2). The control group included 10 nonsmoking patients. Masson's trichrome stain was used to quantify the foreskin vascular density. Weigert’s resorcin-fucsin stain was used to assess the elastic system fibers and Picrosirius red stain was applied to study the collagen. Stereological analysis was performed using the Image J software to determine the volumetric densities. For biochemical analysis, the total collagen was determined as µg of hydroxyproline per mg of dry tissue. Means were compared using the unpaired t-test (p < 0.05). RESULTS: Elastic system fibers of smokers was 42.5% higher than in the control group (p = 0.002). In contrast, smooth muscle fibers (p = 0.42) and vascular density (p = 0.16) did not show any significant variation. Qualitative analysis using Picrosirius red stain with polarized light evidenced the presence of type I and III collagen in the foreskin tissue, without significant difference between the groups. Total collagen concentration also did not differ significantly between smokers and non-smokers (73.1µg/mg ± 8.0 vs. 69.2µg/mg ± 5.9, respectively, p = 0.23). CONCLUSIONS: The foreskin tissue of smoking patients had a significant increase of elastic system fibers. Elastic fibers play an important role in this tissue’s turnover and this high concentration in smokers possibly causes high extensibility of the foreskin. The structural alterations in smokers’ foreskins could possibly explain the poor results in smoking patients submitted to foreskin fasciocutaneous flaps in urethral reconstruction surgery.

Effects of castration and hormone replacement in the urinary bladder of rats: structural, ultrastructural, and biochemical analysis.

We evaluated, by qualitative and quantitative methods, the structural alterations in the bladder wall of rats submitted to surgical castration, as well as the role of hormone replacement in reversing the possible structural alterations. Twenty-four 12-week-old male Sprague-Dawley rats were used. The animals were divided into 3 groups comprising 8 animals each and treated as follows. Members of group CONTR (control) underwent a sham operation only and were sacrificed after 2 months. Members of group ORCH (orchiectomy) underwent bilateral orchiectomy and were sacrificed after 2 months. Members of group ORCH+TEST (testosterone) underwent orchiectomy, received testosterone replacement after 1 month, and were sacrificed 1 month later. We performed a qualitative and quantitative analysis of collagen by light microscopy, scanning electron microscopy, biochemistry, and a histomorphometric analysis of smooth muscle and elastic fibers in the 3 groups. The results showed a significant decrease in absolute values of elastic fibers in the castrated group. The histomorphometric analysis of epithelial height did not show differences among the groups. There was no statistical difference in quantitative analysis of collagen, either by histomorphometry or by biochemistry. Also, there was no difference in the smooth muscle cells. However, the qualitative analysis revealed differences in collagen (castrated group) when compared with controls and with rats submitted to hormone replacement. Hormone replacement with testosterone was able to revert the alterations observed. The findings suggest that hormone replacement, even when instituted at a late stage, is effective in reversing the bladder wall alterations produced by secondary hypogonadism.

Extracellular matrix remodeling in the human gubernaculum during fetal testicular descent and in cryptorchidic children.

PURPOSE: We evaluated extracellular matrix remodeling in human fetal and cryptorchidic gubernacula. METHODS: Gubernacula were obtained from 40 normal human fetuses aged 15-29 weeks postconception (WPC) and from 39 children aged 1.3-10 years who had been submitted to orchiopexy. Total collagen and glycosaminoglycan (GAG) concentrations were assessed as µg hydroxyproline and µg hexuronic acid per mg dry gubernacular tissue, respectively, and proportions of sulfated GAG were determined by agarose gel electrophoresis. RESULTS: Fetal age correlated with collagen (r = 0.77, P < 0.001) and GAG (r = -0.83, P < 0.01) concentrations, which varied from 10 to 50 µg/mg and 7.1-2.5 µg/mg, respectively. Collagen and GAG concentrations in cryptorchidic gubernacula varied from 80.0 to 120.0 µg/mg and from 0.8 to 1.0 µg/mg, respectively. These values did not correlate with patient's age, but when the testis was located more proximally, collagen content was lower. At 15-18 WPC, GAG consisted of 57.3% ± 13.5% chondroitin sulfate and 28.2% ± 10.1% dermatan sulfate, and at 25-28 WPC, 42.7% ± 8.7% and 71.8% ± 11.3%, respectively. GAG in postnatal gubernacula consisted mostly of dermatan sulfate. CONCLUSIONS: From the 15th to the 29th WPC, the extracellular matrix of the gubernaculum undergoes extensive remodeling and this may contribute to testicular descent. Cryptorchidic gubernacula are markedly more fibrous than the corresponding fetal tissue, change little after birth, and have a lower collagen concentration when the undescended testis is abdominal in position.

The penis in diabetes: structural analysis of connective tissue and smooth muscle alterations in a rabbit model.

UNLABELLED: OBJECTIVE • To assess the volumetric density of collagen, elastic system fibres and smooth muscle cells in the corpus cavernosum (CC), corpus spongiosum (CS) and tunica albuginea (TA) in the penis of diabetic rabbits. MATERIALS AND METHODS: • Twenty-six New Zealand white rabbits were used. Diabetes was induced at 8 weeks of age in 13 rabbits by i.v. injection of 100 mg/kg of alloxan. The remaining 13 rabbits served as a control group. After 10 weeks, the rabbits were killed using sodium thiopenthal. • Midshaft penile fragments were obtained and processed by routine histological techniques. Stereological analysis of collagen, elastic system fibres and smooth muscle was performed in 5-µm sections by using a M42 test grid system. • Data were expressed as volumetric density (Vv; %). Collagen organization was evaluated by Picrosirius red staining under polarization. RESULTS: • In the TA of diabetic rabbits, thickness increased by 88% (P < 0.001) with an enhanced collagen turnover. Moreover, the elastic fibre content was 34% higher (P < 0.001). In the CC of diabetics, collagen was diminished by 45% (P < 0.001) with a more organized collagen. • The elastic fibres were decreased by 46% (P < 0.001). Diabetes induced a 11% increase in CS collagen (P < 0.024) with an enhanced collagen turnover. • Smooth muscle in the CC of diabetic rabbits was increased by 40% (P < 0.001), whereas, in the CS, it was decreased by a similar amount (P < 0.001). CONCLUSIONS: • Penile tissues were affected differently by diabetes, possibly as a result of cellular heterogeneity. • These changes could have an impact on blood flow and tissue resistance, and therefore might adversely affect erection.

Platelet-derived growth factor differentially regulates the expression and post-translational modification of versican by arterial smooth muscle cells through distinct protein kinase C and extracellular signal-regulated kinase pathways.

The synthesis of proteoglycans involves steps that regulate both protein and glycosaminoglycan (GAG) synthesis, but it is unclear whether these two pathways are regulated by the same or different signaling pathways. We therefore investigated signaling pathways involved in platelet-derived growth factor (PDGF)-mediated increases in versican core protein and GAG chain synthesis in arterial smooth muscle cells (ASMCs). PDGF treatment of ASMCs resulted in increased versican core protein synthesis and elongation of GAG chains attached to the versican core protein. The effects of PDGF on versican mRNA were blocked by inhibiting either protein kinase C (PKC) or the ERK pathways, whereas the GAG elongation effect of PDGF was blocked by PKC inhibition but not by ERK inhibition. Interestingly, blocking protein synthesis in the presence of cycloheximide abolished the PDGF effect, but not in the presence of xyloside, indicating that GAG synthesis that results from PKC activation is independent from de novo protein synthesis. PDGF also stimulated an increase in the chondroitin-6-sulfate to chondroitin-4-sulfate ratio of GAG chains on versican, and this effect was blocked by PKC inhibitors. These data show that PKC activation is sufficient to cause GAG chain elongation, but both PKC and ERK activation are required for versican mRNA core protein expression. These results indicate that different signaling pathways control different aspects of PDGF-stimulated versican biosynthesis by ASMCs. These data will be useful in designing strategies to interfere with the synthesis of this proteoglycan in various disease states.

Oral hormonal contraceptives affect the concentration and composition of urinary glycosaminoglycans in young women.

INTRODUCTION AND HYPOTHESIS: Urinary glycosaminoglycans (uGAG) have protective effects against urinary tract disorders. Here we investigated whether oral hormonal contraceptives (OC) affect uGAG excretion. METHODS: Urine specimens were from young women regularly taking: ethinyl estradiol + drospirenone, n = 9; ethinyl estradiol + cyproterone acetate, n = 9; and ethinyl estradiol + gestodene, n = 7. Controls were from ten women not taking OC. Total uGAG was assayed as hexuronic acid/urinary creatinine. Sulfated uGAG species was determined by electrophoresis. RESULTS: Unlike controls, total uGAG in the two halves of the menstrual cycle was similar in the OC groups. Whole cycle uGAG was higher in the OC groups (p < 0.01), especially for ethinyl estradiol + cyproterone acetate (p < 0.005). The three OC produced decreases of approximately 50% in heparan sulfate (p < 0.02) and dermatan sulfate (p < 0.02), and a approximately 100% increase in chondroitin sulfate (p < 0.004). CONCLUSIONS: uGAG excretion is changed in women taking OC, and this might enhance the protective effects of these molecules against urinary tract disorders.

Stereological and biochemical analysis of muscular and connective tissue components in the penile corpus cavernosum adjacent to the fibrous plaque of Peyronie's disease.

OBJECTIVE: To investigate the structural organization of the connective tissue in the corpus cavernosum (CC) adjacent to the fibrous plaque in Peyronie's disease (PD) using stereological and biochemical techniques, as most studies on PD have focused on the analysis of the fibrous plaque that forms in the tunica albuginea (TA). Because this fibrotic reaction is mediated by various inflammatory soluble factors, adjacent connective tissues might also be affected and this secondary effect might explain, for example, the erectile dysfunction that occurs in PD. PATIENTS AND METHODS: During surgery biopsies were taken from the CC adjacent to the fibrous plaque and from the plaque itself in seven patients with PD (mean age 48.3 years). All the patients had normal erections. Control samples were similarly located samples from 'normal' penises obtained during autopsy of five men (mean age 52.3 years). Tissue samples were stained with Weigert's stain (elastic fibres), Van Gieson's stain (connective tissue), and Sirius red (collagen). Stereological analysis was done using a 42-point grid to determine volumetric densities (Vv). Total collagen content was estimated as micrograms of hydroxyproline per milligram dry CC. RESULTS: The Vv of elastic fibres was significantly reduced in PD by 17.3% compared with controls, at a mean (sd) of 19.49 (3.27)% vs 23.56 (1.87)% (P < 0.05). While in PD the Vv of smooth muscle at 34.46 (2.06)% and connective tissue at 35.39 (6.15)% were not significantly different from those of controls at 38.38 (3.17)% and 38.02 (5.03)%, respectively. The Vv of elastic fibres in the fibrous plaque was decreased by 38.3% compared with the normal TA, at 20.25 (5.49)% vs 32.81 (4.75)% (P < 0.02). The mean (sd) collagen concentration in the CC from controls was 77.94 (24.26) microg/mg and in the patients with PD was 66.57 (19.39) microg/mg, which did not differ significantly. Sirius red-stained sections under polarized light showed that, in the normal CC, collagen-associated colours were homogeneously distributed. However, in the PD samples, stained collagen had a disrupted orientation and had a more heterogeneous birefringence, implying looser collagen bundles. CONCLUSIONS: The quantitative analyses indicated that collagen in the CC close to the fibrous plaque was not affected, although its organization was noticeably altered. The CC elastic fibres were reduced though, and there was a similar change in the fibrous plaque of the TA. These results suggest that, although occurring primarily in the TA, the PD fibrous plaque may induce changes in the adjacent CC.