New in vivo model to assess macroscopic, histological, and molecular changes in Peyronie's disease.

BACKGROUND: Peyronie's Disease (PD) is a connective tissue disorder that affects the tunica albuginea (TA) of the penis causing curvature and erectile dysfunction. The pathophysiology is not well understood and, for this reason, treatment options are limited. OBJECTIVE: The aim of the present study is to analyze and compare whether single or multiple instillations of plasma in the TA of rats is capable of triggering macroscopic, histopathological, and molecular changes consistent with PD. MATERIAL/METHODS: Fifty male Wistar rats were divided into four groups: Group 1: a single instillation of plasma in the TA; Group 2: a single instillation of distilled water in the TA; Group 3: four instillations of plasma in the TA (1x per week); and Group 4: four instillations of distilled water in the TA (1× per week). Forty-five days after the last instillation a manual inspection of the corpus cavernosum, a penile erection test and a penectomy were performed to obtain material for histopathological and molecular analysis. RESULTS: It was observed that 31.25% of the rats that received repeated instillations of plasma presented penile curvature according to the erection test, while none of the rats from the control group or group with one instillation of plasma presented curvature. In the animals that received four instillations of plasma, the following differences were observed in relation to the control group: increase in fibrosis and the deposition of collagen I. The protein expression of heparanase (HPSE) and TGF-ß increased in the groups that received a single or four instillations of plasma, and the protein expression of heparanase-2 (HPSE-2), metalloproteinases (MMP-2, MMP-9) and metalloproteinase inhibitor (TIMP-2) showed an increase in the group that received four instillations of plasma. There was a significant increase in the gene expression of HPSE, MMP-9, and TGF-ß in the group that received four instillations of plasma. In the analysis of the glycosaminoglycans, an increase was observed in the secretion of galactosaminoglycans chondroitin sulfate and dermatan sulfate (CS/DS) in the group that received four instillations of plasma. DISCUSSION: Previous studies have demonstrated increased protein expression. of HPSE, MMP-9 and TGF-ß with instillation of blood in the TA; however, there was no increase in gene expression. In the present study, the increase in the expression of TGF-ß with plasma instillations, proved to be more reliable. The two models with plasma (one or four instillations) demonstrated significant histopathological and molecular changes when compared to the control group. However, only in the group with four plasma instillations there was a macroscopic change. The idea is that repeatedly extravasation of TGF-ß present in plasma of predisposed individuals acts as a trigger for the development and maintenance of changes in the extracellular matrix that perpetuate an anomalous inflammatory process present in PD. CONCLUSION: The present study shows that the repeated instillation of plasma is a low cost in vivo model for the study of PD.

Endocytosis and the Participation of Glycosaminoglycans Are Important to the Mechanism of Cell Death Induced by ß-Hairpin Antimicrobial Peptides.

The cytotoxic mode of action of four antimicrobial peptides (AMPs) (gomesin, tachyplesin, protegrin, and polyphemusin) against a HeLa cell tumor model is discussed. A study of cell death by AMP stimulation revealed some similarities, including annexin-V externalization, reduction of mitochondrial potential, insensitivity against inhibitors of cell death, and membrane permeabilization. Evaluation of signaling proteins and gene expression that control cell death revealed wide variation in the responses to AMPs. However, the ability to cross cell membranes emerged as an important characteristic of AMP-dependent cell death, where endocytosis mediated by dynamin is a common mechanism. Furthermore, the affinity between AMPs and glycosaminoglycans (GAGs) and GAG participation in the cytotoxicity of AMPs were verified. The results show that, despite their primary and secondary structure homology, these peptides present different modes of action, but endocytosis and GAG participation are an important and common mechanism of cytotoxicity for ß-hairpin peptides.

Type II Diabetes Patients under Sildenafil Citrate: Case Series Showing Benefits and a Side Effect.

BACKGROUND: Diabetes mellitus is a highly prevalent disease with rapid universal growth. In 2013, there were already 382 million people with diabetes, and it is expected that by 2035, this number will double. Chronic hyperglycemia causes a series of biochemical and structural changes, especially in the eyes, kidneys, heart, arteries, and peripheral nerves, which usually leads to the progression of microvascular disease. Several literature reports showed that the chronic use of phosphodiesterase-5 inhibitors enhances the insulin sensitivity, improves the markers of endothelial function, and helps in the treatment of severe extremity ischemia and pulmonary hypertension. We aim to test the effect of sildenafil citrate (SC) as a glucose and microcirculation regulator in diabetic patients, paying special attention to the consequences of its use in the regulation of blood glucose level. Case Presentation. Two male patients, aged 53 and 73 years, with type II diabetes, using oral hypoglycemic agents and presenting pathology associated with microcirculation alterations and ischemia, were medicated daily with SC. Both patients presented a reduction in the glycemic level, requiring lower doses or no other oral diabetes medications. Patient 1, who presented diabetic foot, was treated in the ambulatory, and patient 2, with chronic obstructive pulmonary disease and consequent mild pulmonary hypertension, was treated in the office. In addition to the clinical improvement of foot wounds and dyspnea due to the increase in microcirculatory perfusion, hypoglycemic episodes were observed in both patients under SC. The patient with pulmonary hypertension experienced one severe hypoglycemia episode and had to be taken to an emergency room. CONCLUSION: Type 2 diabetic patients may benefit from the use of a phosphodiesterase-5 inhibitor in order to improve the microcirculatory perfusion as well as glycemic control. However, adverse side effects may involve hypoglycemia. Since off-label use of SC in patients suffering from microcirculatory alterations has increased recently, our results showed that more studies are needed to verify the prevalence of hypoglycemia episodes as well as it's possible physiologic mechanism.

Distribution of heparan sulfate correlated with the expression of heparanase-1 and matrix metalloproteinase-9 in an ovariectomized rats skin.

There are few studies on heparan sulfate (HS) in the skin, during aging, when estrogen is suppressed. The enzyme heparanase-1 (HPSE-1), has its 17ß-estrogen-regulated expression in pathological conditions such as cancer and chronic inflammatory diseases. HPSE-1 is correlated with the matrix metalloproteinase-9 (MMP-9), an endopeptidase that also undergoes estrogen action. We investigated the distribution of HS, expression HPSE-1 and MMP-9 in the skin of adult rats at different ages and in the age-matched ovariectomized rats to evaluate the influence of low estrogen on the distribution of HS. Thirty female Wistar rats were used. Rats underwent to a sham surgery (ctr, n = 15) or to a bilateral ovariectomy (ovx, n = 15) and were euthanized after 45, 75, and 90 days after ovariectomy. Morphological, morphometric, biochemical, and reverse transcriptase polymerase chain reaction (RT-PCR) methodologies were used. A significant decrease (P < 0.001) in total skin thickness was observed in the ctr and ovx animals, being higher in the older animals. The thickness of the epidermis and dermis decreased; however, the proportion in the total skin remained similar comparing ctr and ovx. An increase of HS with increasing age and ovariectomy was observed. The expression of the HPSE-1 and MMP-9 enzymes decreased, being higher in old animals. A correlation between the increase of HS and the decrease of the HPSE-1 was demonstrated in both groups. Overall, these data suggested that estrogen acts in the regulation of the expression of the HPSE-1, not only in pathological states, as already established, but also in aging.

Molecular alterations of human lumbar yellow ligament related to the process of intervertebral disk degeneration and stenosis.

PURPOSE: The objective of this study was to analyze the layers of yellow ligament in lumbar canal stenosis and disk herniation. METHODS: Eighteen ligaments were harvested from patients with lumbar spinal canal stenosis. Twenty-nine normal samples from lumbar spine disk herniation patients served as control. All surgical procedures were the same. Ligaments were stained in hematoxylin and eosin; picrosirius-hematoxylin for collagen; Weigert's resorcin-fuchsin for elaunin, oxytalan and elastic fibers; and transmission electron microscopy. Immunohistochemistry was performed for Il-6; Il-10; and CD-31, PGP9.5. Results are described in means and standard error (mean ± SE), and all analyses adopted the significance level of P < 0.05. RESULTS: Spinal stenosis ligaments were 2.5 × thicker. Control superficial ligaments presented a large number of thick, compact collagen fibers and a significant amount of oxytalan and mature elastic fibers. The deep layer presented a large number of mature elastic fibers. In the stenosis group, collagen was thinner and compacted in both layers. There was no difference in the interleukin profile among groups. The deep portion of the stenosis group presented a higher number of vessels and nerves. CONCLUSION: Two layers compose the elastic system of the normal ligamentum flavum, where the deep portion is mainly responsible for its elasticity (elaunin fibers), while its resistance depends on the concentration of oxytalan fibers, which are more present in the superficial layer. Ligamentum flavum in the stenosis samples presents more mononuclear infiltrate and more degraded elastic fibers with a higher number of vessels in its deep portion. These slides can be retrieved under Electronic Supplementary Material.

Extracellular matrix alterations after blood instillation in tunica albuginea of rats.

The cause of Peyronie's disease (PD) is still not completely understood. The objective of this study, therefore, was to analyze the histological and biochemical alterations that occur after the instillation of blood in the tunica albuginea (TA) of rats with an emphasis on the remodeling process of ECM. Thirty male Wistar rats were divided into 4 groups: two control groups with instillation of distilled water in TA followed by penectomy after 15 days or 45 days, respectively and two experimental groups with instillation of blood in TA followed by penectomy after 15 days or 45 days, respectively. Histological, immunofluorescent and immunohistochemical analyses were performed. The higher presence of fibrotic tissue in rats injected with blood demonstrated alterations in TA similar to inflammation found in PD. The increased expression of TGF-ß, MMP9, HPSE, and biglycan associated with the decreased expression of syndecan-1 and aggrecan in the experimental groups suggested an enhancement in the remodeling of ECM. The results contribute to show that blood instillation on TA appears to trigger alterations in the ECM similar to the ones found in inflammatory diseases such as PD.

Glycosaminoglycans affect heparanase location in CHO cell lines.

Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-ß-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [(35)S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.

On the catalytic mechanism of polysaccharide lyases: evidence of His and Tyr involvement in heparin lysis by heparinase I and the role of Ca2+.

The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis. We demonstrate that heparinase I specificity and efficiency depend on the cationic form of the substrate. Ca(2+)-heparin, in which α-L-iduronate-2-O-sulfate residues adopt (1)C4 conformation preferentially, is a substrate, while Na(+)-heparin is an inhibitor. His and Tyr residues are identified in the catalytic step and a model based on molecular dynamics and docking is proposed, in which deprotonated His203 initiates ß-elimination by abstracting the C5 proton of the α-L-iduonate-2-O-sulfate residue in the substrate, and protonated Tyr357 provides the donor to the hexosamine leaving group.

Colorectal cancer desmoplastic reaction up-regulates collagen synthesis and restricts cancer cell invasion.

During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.

Inhibitory peptides of the sulfotransferase domain of the heparan sulfate enzyme, N-deacetylase-N-sulfotransferase-1.

N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate and heparin during their biosynthesis by removal of acetyl groups from subsets of N-acetylglucosamine units and subsequent sulfation of the resulting free amino groups. In this study, we used a phage display library to select peptides that interact with Ndst1, with the aim of finding inhibitors of the enzyme. The phage library consisted of cyclic random 10-mer peptides expressed in the phage capsid protein pIII. Selection was based on the ability of engineered phage to bind to recombinant murine Ndst1 (mNdst1) and displacement with heparin. Peptides that were enriched through multiple cycles of binding and disassociation displayed two specific sequences, CRGWRGEKIGNC and CNMQALSMPVTC. Both peptides inhibited mNdst1 activity in vitro, however, by distinct mechanisms. The peptide CRGWRGEKIGNC presents a chemokine-like repeat motif (BXX, where B represents a basic amino acid and X is a noncharged amino acid) and binds to heparan sulfate, thus blocking the binding of substrate to the enzyme. The peptide NMQALSMPVT inhibits mNdst1 activity by direct interaction with the enzyme near the active site. The discovery of inhibitory peptides in this way suggests a method for developing peptide inhibitors of heparan sulfate biosynthesis.

The binding of heparin to the extracellular matrix of endothelial cells up-regulates the synthesis of an antithrombotic heparan sulfate proteoglycan.

Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS. In kinetic studies the BiotHep showed fast, saturable and specific binding with an apparent K(D) of 83 nM to adherent cells and 44 nM to the extracellular matrix (ECM) in the absence of cells. By confocal and electron microscopy, BiotHep bound only to the ECM, co-localizing with fibronectin. The same pattern of binding to the ECM was observed using heparin conjugated with FITC or Alexa Fluor 488 in the presence or absence of fetal calf serum. However, after degradation of HS, heparin binds to the cell surface, indicating that endogenous HS possibly occupied the heparin binding sites. Analyses by flow cytometry and confocal microscopy of cells with non-associated ECM, showed labeling of the cell surface using syndecan-4 monoclonal antibody as well as wheat germ agglutinin, but no binding of heparin. Furthermore, the stimulation in HS synthesis is not elicited by heparin in the absence of ECM. These results indicate that the stimulus for the synthesis of HS does not require binding of the heparin to the cell surface, and the signaling may be mediated through the ECM.

Melanocyte transformation associated with substrate adhesion impediment.

Exclude experimental models of malignant transformation employ chemical and physical carcinogens or genetic manipulations to study tumor progression. In this work, different melanoma cell lines were established after submitting a nontumorigenic melanocyte lineage (melan-a) to sequential cycles of forced anchorage impediment. The great majority of these cells underwent anoikis when maintained in suspension. After one deadhesion cycle, phenotypic alterations were noticeable in the few surviving cells, which became more numerous and showed progressive alterations after each adhesion impediment step. No significant differences in cell surface expression of integrins were detected, but a clear electrophoretic migration shift, compatible with an altered glycosylation pattern, was observed for beta1 chain in transformed cell lines. In parallel, a progressive enrichment of tri- and tetra-antennary N-glycans was apparent, suggesting increased N-acetylglucosaminyltransferase V activity. Alterations both in proteoglycan glycosylation pattern and core protein expression were detected during the transformation process. In conclusion, this model corroborates the role of adhesion state as a promoting agent in transformation process and demonstrates that cell adhesion disturbances may act as carcinogenic stimuli, at least for a nontumorigenic immortalized melanocyte lineage. These findings have intriguing implications for in vivo carcinogenesis, suggesting that anchorage independence may precede, and contribute to, neoplastic conversion.