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Chronic wounds are a serious healthcare problem. As non-healing wounds involve continuous pathologic inflammatory stage, research is focused on anti-inflammatory treatments. Our objective was to analyze the effect of S42909, a potent NADPH oxidase inhibitor activity, with vascular anti-inflammatory properties. An ischemic rabbit ear ulcer model (24 New Zealand white rabbits) was used to evaluate the reepithelialization/contraction areas, anti-/pro-inflammatory cytokines mRNA (TGF-β1/IL-10/IFN-γ/VEGF) by qRT-PCR, collagen I/III deposition, and neovascularization (TGF-β1/VEGF) by morphological and immunohistochemical analyses. Three different doses were administered by gavage for 2 weeks: 10 and 30 mg/kg/d in self-microemulsion drug delivery system (SMEDDS) and 100 mg/kg/d in arabic gum. Each vehicle was used as control. No signs of infection or necrosis were found. Reepithelialization was almost complete whatever the groups reaching 95% at the dose of 100 mg/kg. Wound contraction was significantly reduced in all S42909-treated groups. A significant increase in anti-inflammatory cytokines TGF-β1 mRNA and IL-10 mRNA was observed at the dose of 100 and 30 mg/kg/d, respectively. No changes were observed in pro-inflammatory factors INF-γ and VEGF mRNA. Ischemic skin wound areas had scarce expression of collagen I/III and showed rich glycosaminoglycans content. Treatment increased the collagen deposition and TGF-β1 protein expression and decreased glycosaminoglycan content dose dependently; however, no effect in VEGF was appreciated. Therefore, our results indicate that S42909 improved healing process by dampening excessive inflammation and facilitating collagen deposition without wound contraction phenomena. S42909 might be a promising therapy to treat chronic wounds as venous leg ulcers., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aims: This study examines the intraperitoneal behavior of two cyanoacrylate tissue adhesives: Ifabond ® and a new, non-marketed octyl cyanoacrylate adhesive (OCA) used for the intraperitoneal fixation of a laminar expanded polytetrafluoroethylene (ePTFE) mesh., Material and Methods: In 36 New Zealand White rabbits, 3 × 3 cm (n = 24) or 1.5 × 3 cm (n = 12) fragments of ePTFE mesh (Preclude ® , Gore, Flagstaff, USA) were fixed to the parietal peritoneum using OCA or Ifabond ® . Peritoneal fluid was obtained at the time of implant and at 2 weeks postimplant for determination of the cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). At 14 or 90 days postsurgery, the animals were euthanized and the meshes excised to assess host tissue incorporation, the macrophage response, apoptosis and fixation strength (T-peel tensiometry)., Results: Peritoneal fluid IL-6 and TNF-α concentrations were similar in the OCA and Ifabond ® groups. Both adhesives gave rise to adequate mesothelialization of the laminar ePTFE. Macrophage counts were similar for the two study groups, but a significantly increase in macrophage response was observed from 14 to 90 days for Ifabond ® . At 90 days postimplant, apoptotic cell counts was lower for the implants fixed with OCA and a fixation strength was significantly lower for OCA., Conclusions: Despite similar cytokine levels at 2 weeks and similar host tissue incorporation observed for the meshes fixed with the two adhesives, the use of Ifabond ® gave rise to a greater apoptosis rate, although this adhesive provided a stronger fixation bond. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 312-319, 2017., (© 2015 Wiley Periodicals, Inc.)

Background: Cyanoacrylate(CA)-based tissue adhesives, although not widely used, are a feasible option to fix a mesh during abdominal hernia repair, due to its fast action and great bond strength. Their main disadvantage, toxicity, can be mitigated by increasing the length of their alkyl chain. The objective was to assess the in vitro cytotoxicity and in vivo biocompatibility in hernia repair of CAs currently used in clinical practice (Glubran(n-butyl) and Ifabond(n-hexyl)) and a longer-chain CA (OCA(n-octyl)), that has never been used in the medical field., Methods: Formaldehyde release and cytotoxicity of unpolymerized(UCAs) and polymerized CAs(PCAs) were evaluated by macroscopic visual assessment, flow cytometry and Alamar Blue assays. In the preclinical evaluation, partial defects were created in the rabbit abdominal wall and repaired by fixing polypropylene prostheses using the CAs. At 14 days post-surgery, animals were euthanized for morphology, macrophage response and cell damage analyses., Results: Formaldehyde release was lower as the molecular weight of the monomer increased. The longest side-chain CA(OCA) showed the highest cytotoxicity in the UCA condition. However, after polymerization, was the one that showed better behavior on most occasions. In vivo, all CAs promoted optimal mesh fixation without displacements or detachments. Seroma was evident with the use of Glubran, (four of six animals: 4/6) and Ifabond (2/6), but it was reduced with the use of OCA (1/6). Significantly greater macrophage responses were observed in groups where Glubran and Ifabond were used vs. sutures and OCA. TUNEL-positive cells were significantly higher in the Glubran and OCA groups vs. the suture group., Conclusions: Although mild formaldehyde release occurred, OCA was the most cytotoxic during polymerization but the least once cured. The CAs promoted proper mesh fixation and have potential to replace traditional suturing techniques in hernia repair; the CAs exhibited good tissue integration and effective short-term biocompatibility, with the slightest seroma and macrophage response induced by OCA.

Background: Bioprostheses represent a significant advance in the abdominal wall reconstruction since they become degraded until their complete elimination in the recipient organism. This study examines remodeling in the host of three noncrosslinked porcine dermal collagen biomeshes: Strattice™ (St; LifeCell Corp.), XCM Biologic® Tissue Matrix (XCM; Synthes CMF) and Protexa® (Pr; Deco Med S.R.L.)., Methods: Partial ventral hernia defects created in New Zealand White rabbits were repaired using the biomeshes that were placed in an inlay, preperitoneal position. At 14 and 90 days after implantation, explants were assessed in terms of their host tissue incorporation by morphological studies, collagen gene/protein expression (quantitative real-time PCR/immunofluorescence), macrophage response (immunohistochemistry) and biomechanical strength., Results: There were no cases of mortality or infection. Among our macroscopic findings, the mesh detachment detected in one third of the Pr implants at 90 days was of note. The host tissue response to all the biomeshes was similar at both time points, with a tendency observed for their encapsulation. There were no appreciable signs of mesh degradation. The extent of host tissue infiltration and collagenization was greater for St and Pr than for XCM. Macrophages were observed in zones of inflammation and tissue infiltration inside the mesh. XCM showed a greater macrophage response at 90 days (p < 0.05). Improved tensile strength was observed for St (p < 0.05) over Pr and unrepaired defects., Conclusions: St showed the best behavior, featuring good collagenization and tensile strength while also inducing a minimal foreign body reaction., (© 2015 S. Karger AG, Basel.)

Background: The use of a prosthetic material is the best treatment option for ventral hernia repair; one of the most frequently performed abdominal surgery procedures. This preclinical study compares the behavior of a new mesh (Parietex composite ventral patch [Ptx]) with that of two existing meshes used for ventral hernia repair., Materials and Methods: Fifty-four New Zealand White rabbits (3000 g) were used in an experimental model of umbilical hernia repair (diameter 1.5 cm). The materials tested were: Ventralex ST hernia patch (Vent) (Bard Davol Inc, Warwick, RI) (n = 18); Proceed ventral patch (Ethicon, Somerville, NJ) (PVP) (n = 18) and Ptx (Covidien, Sofradim, Trevoux, France) (n = 18). At 3, 7, 14 d, and 6 wk after implant, peritoneal behavior and adhesion formation were assessed by sequential laparoscopy. Mesh mesothelial cover was determined by scanning electron microscopy. Host tissue ingrowth (collagens I and III) and the macrophage response were assessed by immunohistochemical labeling. Animals were euthanized at 2, 6 wk, and 6 mo after surgery. Data were compared using the Mann-Whitney U test., Results: Adhesion formation from 3 d-6 wk was significantly greater (P < 0.05) for PVP compared with Vent or Ptx. Three encapsulated PVP implants showed "tissue-integrated" adhesions affecting the intestinal loops. All three implant types showed similar patterns of collagen l and III deposition. The PVP mesh elicited the greater macrophage response both at 2 wk and 6 mo., Conclusions: Ptx and Vent showed excellent mesothelialization, which led to minimum adhesion formation. The appropriate tissue integration of Ptx in the parietal neoperitoneum is likely attributable to its deployment system., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Midline laparotomy closure carries a significant risk of incisional hernia. This study examines the behavior of two new suture materials, an elastic material, polyurethane (PUe), and a barbed polydioxanone (PDXb) suture thread in a rabbit model of midline incision closure., Methods: Three 2-cm midline incisions were made in 68 New Zealand White rabbits. The incisions were closed by running suture using four 3/0 threads: polypropylene (PP) (Surgipro®, Covidien), PUe (Assuplus®, Assut Europe), PDX (Assufil®, Assut Europe) or PDXb (Filbloc®, Assut Europe). Animals in each suture group were euthanized 3 weeks and 6 months after surgery. Histological sections of the tissue-embedded sutures were subjected to morphological, collagen expression, macrophage response and uniaxial tensiometry studies., Results: No signs of wound dehiscence or complications were observed. At 3 weeks, all sutures were surrounded by connective tissue composed mainly of collagen III. PUe showed greater collagen I expression than the other sutures. All sutures elicited a macrophage response that diminished from 3 weeks to 6 months (p < 0.001). This response was similar for the non-reabsorbable sutures (PP and PUe) yet PDXb showed a significantly greater response than the other reabsorbable suture (PDX) at 3 weeks (p < 0.01). At this early time point, the tensile strength of PUe was similar to that of control intact tissue (p > 0.05)., Conclusion: Three weeks after surgery, PUe revealed more collagen I deposition than the remaining materials and this translated to a similar biomechanical behavior to linea alba, that could avoid the appearance of short term dehiscences and thus reduce the incidence of incisional hernia. PDXb provides no additional advantages in their behavior regarding PDX suture.

Purpose: We evaluated the expression of several extracellular matrix constituents implicated in the synthesis and reticulation of elastin in human pterygium, according to age and sex of the patients., Methods: Pterygia and normal conjunctiva samples were divided into groups according to age (<50/≥50 years) and sex. Tissue was subjected to immunohistochemical staining with anti-lysyl oxidase (LOX), lyxyl oxidase-like 1 (LOXL-1), fibulin (FBLN)-5 and FBLN4, and fibrillin-1 (FBN1) antibodies. Specific primers for the same constituents were used in a quantitative real-time PCR (qRT-PCR) analysis to determine gene expression., Results: The LOXL-1 (P = 0.0002), FBLN5 (P = 0.0035), and FBN1 (P < 0.0001) mRNAs were significantly higher in pterygium than conjunctiva. No differences were found for LOX and FBLN4 mRNA. The expression of LOXL-1 was not affected by age or sex; however, pterygium from men and patients over 50 years old exhibited significantly higher FBLN5/FBN1 expression than did controls. The LOX gene expression was higher in the pathologic samples from the over 50-year-olds compared to the conjunctiva (P = 0.0396) and in men's versus women's pterygium (P = 0.0173). In general, the levels of LOX, LOXL-1, and FBLN5 decreased with age in healthy samples, while the pathology seemed to have increased expression of the three proteins, and even more so in older patients. The FBLN4 and FBN1 immunostaining was slight in all samples, displaying no differences between groups., Conclusions: Several extracellular matrix constituents, LOXs, FBN1, and FBLN5, implicated in the development of elastin, are overexpressed in the subepithelial connective tissue extracellular matrix of human pterygium, supporting our hypothesis that elastic synthesis and reticulation is dysregulated in this type of pathology., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

This study explores the importance of different motivations to visit three types of recreational settings--farms, private forests, and state or national parks. Data were collected via a mail-back questionnaire administered to a stratified random sample of households in Missouri (USA). Descriptive and inferential statistics reveal both similarities and discontinuities in motivations for visiting farms, private forests, and state or national parks for recreation. Being with family, viewing natural scenery, and enjoying the smells and sounds of nature were all highly important motivations for visiting the three types of settings. However, all 15 motivations examined were perceived to be significantly more important for visits to state or national parks than to farms or private forests. Findings suggest that individuals are more strongly motivated to recreate at state and national parks relative to farmlands or forests. Post hoc paired t tests comparing motivations between both agricultural settings (farms and private forests) revealed significant differences in eight different recreational motivations. Individuals tended to place more importance on the ability to use equipment and test their skills when considering recreating on private forests. Conversely, social motivations (e.g., doing something with the family) were more important when individuals were considering recreating on farmland. Collectively, the findings suggest individuals expect distinctly different outcomes from their visits to farmlands, private forests, or state or national parks. Consequently, all three types of recreational settings have competitive advantages that their managers could capitalize on when making decisions about how to attract new visitors or produce the most desirable experiences for current recreationists.

Introduction: Composite biomaterials designed for the repair of abdominal wall defects are composed of a mesh component and a laminar barrier in contact with the visceral peritoneum. This study assesses the behaviour of a new composite mesh by comparing it with two latest-generation composites currently used in clinical practice., Methods: Defects (7x5cm) created in the anterior abdominal wall of New Zealand White rabbits were repaired using a polypropylene mesh and the composites: Physiomesh(TM); Ventralight(TM) and a new composite mesh with a three-dimensional macroporous polyester structure and an oxidized collagen/chitosan barrier. Animals were sacrificed on days 14 and 90 postimplant. Specimens were processed to determine host tissue incorporation, gene/protein expression of neo-collagens (RT-PCR/immunofluorescence), macrophage response (RAM-11-immunolabelling) and biomechanical resistance. On postoperative days 7/14, each animal was examined laparoscopically to quantify adhesions between the visceral peritoneum and implant., Results: The new composite mesh showed the lowest incidence of seroma in the short term. At each time point, the mesh surface covered with adhesions was greater in controls than composites. By day 14, the implants were fully infiltrated by a loose connective tissue that became denser over time. At 90 days, the peritoneal mesh surface was lined with a stable mesothelium. The new composite mesh induced more rapid tissue maturation than Physiomesh(TM), giving rise to a neoformed tissue containing more type I collagen. In Ventralight(TM) the macrophage reaction was intense and significantly greater than the other composites at both follow-up times. Tensile strengths were similar for each biomaterial., Conclusions: All composites showed optimal peritoneal behaviour, inducing good peritoneal regeneration and scarce postoperative adhesion formation. A greater foreign body reaction was observed for Ventralight(TM). All composites induced good collagen deposition accompanied by optimal tensile strength. The three-dimensional macroporous structure of the new composite mesh may promote rapid tissue regeneration within the mesh.

Background: Although heavyweight (HW) or lightweight (LW) polypropylene (PP) meshes are widely used for hernia repair, other alternatives have recently appeared. They have the same large-pore structure yet are composed of polytetrafluoroethylene (PTFE). This study compares the long-term (3 and 6 months) behavior of meshes of different pore size (HW compared with LW) and composition (PP compared with PTFE)., Methods: Partial defects were created in the lateral wall of the abdomen in New Zealand White rabbits and then repaired by the use of a HW or LW PP mesh or a new monofilament, large-pore PTFE mesh (Infinit). At 90 and 180 days after implantation, tissue incorporation, gene and protein expression of neocollagens (reverse transcription-polymerase chain reaction/immunofluorescence), macrophage response (immunohistochemistry), and biomechanical strength were determined. Shrinkage was measured at 90 days., Results: All three meshes induced good host tissue ingrowth, yet the macrophage response was significantly greater in the PTFE implants (P < .05). Collagen 1/3 mRNA levels failed to vary at 90 days yet in the longer term, the LW meshes showed the reduced genetic expression of both collagens (P < .05) accompanied by increased neocollagen deposition, indicating more efficient mRNA translation. After 90-180 days of implant, tensile strengths and elastic modulus values were similar for all 3 implants (P > .05)., Conclusion: Host collagen deposition is mesh pore size dependent whereas the macrophage response induced is composition dependent with a greater response shown by PTFE. In the long term, macroporous meshes show comparable biomechanical behavior regardless of their pore size or composition., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Collagen prostheses used to repair abdominal wall defects, depending on their pretreatment (noncross-linked vs. cross-linked), besides repair may also achieve tissue regeneration. We assessed the host tissue incorporation of different bioprostheses using a new tool that combines immunofluorescence confocal microscopy with differential interference contrast images, making it possible to distinguish newly formed collagen. Partial hernial defects in the abdominal wall of rabbits were repaired using cross-linked/noncross-linked bioprostheses. Expanded polytetrafluoroethylene (ePTFE) was used as control. After 14/30/90/180 days of implant, specimens were taken for microscopy, immunohistochemistry, and quantitative-reverse transcription-polymerase chain reaction to determine host tissue ingrowth and collagen I/III protein and 1a1/3a1 gene expression. Shrinkage and stress resistance were also examined. At 14 days, cross-linked prostheses had suffered significantly less shrinkage than ePTFE or noncross-linked prostheses. Significantly higher shrinkage was recorded for ePTFE in the longer term. Microscopy revealed encapsulation of ePTFE by neoformed tissue, while the bioprostheses became gradually infiltrated by host tissue. Noncross-linked prosthesis showed better tissue ingrowth, more intense inflammatory reaction and more rapid degradation than the cross-linked prostheses. At 14 days, cross-linked prostheses induced up-regulated collagen 1a1 and 3a1 gene expression, while noncross-linked only showed increased collagen III protein expression at 90 days postimplant. At 6 months, the tensile strengths of cross-linked prostheses were significantly greater compared with ePTFE. Our findings demonstrate that despite the cross-linked collagen prostheses promoting less tissue ingrowth than the noncross-linked meshes, they became gradually replaced by good quality host tissue and were less rapidly degraded, leading to improved stress resistance in the long term., (© 2012 by the Wound Healing Society.)

Introduction: Biological and synthetic laminar absorbable prostheses are available for the repair of hernia defects in the abdominal wall. They share the important feature of being gradually degraded in the host, resulting in place the formation of a neotissue. This study was designed to assess the host tissue's incorporation of collagen bioprostheses and a synthetic absorbable prosthesis., Methods: Partial defects were created in the abdominal walls of 72 New Zealand rabbits and repaired using collagen bioprostheses Tutomesh® and Strattice® or a synthetic prosthesis Bio-A®. Specimens were collected for light microscopy, collagens gene and protein expression, macrophage response and biomechanical resistance at 14, 30, 90 and 180 days post-implantation., Results: Tutomesh® and Bio-A® were gradually infiltrated by the host tissue and almost completely degraded by 180 days post-implantation. In contrast, Strattice® exhibited material encapsulation, no prosthetic degradation and low cell infiltration at earlier timepoints, whereas at later study time, collagen deposition could be observed within the mesh. In the short term, Bio-A® exhibited higher level of collagen 1 and 3 mRNA expression compared with the two other biological prostheses, which exhibited two peaks of higher expression at 14 and 90 days. The expression of collagen III was homogeneous throughout the study and collagen I deposition was more evident in Strattice®. Macrophage response decreased over time in biomeshes. However, in the synthetic mesh remained high and homogeneous until 90 days. The biomechanical analysis demonstrated the progressively increasing tensile strength of all biomaterials., Conclusions: The tissue infiltration of laminar absorbable prostheses is affected by the structure and composition of the mesh. The synthetic prosthesis exhibited a distinct pattern of tissue incorporation and a greater macrophage response than did the biological prostheses. Of all of the laminar, absorbable biomaterials that were tested in this study, Strattice® demonstrated the optimal levels of integration and degradation.

Background: When repairing an abdominal wall defect, sometimes a prosthetic mesh needs to be placed directly on the parietal peritoneum. Although the standard mesh for this purpose is the laminar implant expanded polytetrafluoroethylene (PTFE), it is gradually being replaced by the laminar collagen-based meshes. This study was designed to assess the intraperitoneal behavior of three of these biomeshes, mainly in terms of their susceptibility to adhesion formation., Methods: Two 3-cm × 3-cm fragments of prosthetic material were placed on the parietal peritoneum in male New Zealand White rabbits in the following combinations: PTFE and CollaMend(®), PTFE and Permacol(®), or PTFE and Surgisis(®). The meshes were fixed at the four corners with individual 4/0 polypropylene sutures. Adhesion formation was quantified by sequential laparoscopy and image analysis performed at 3, 7, 14, and 90 days postimplant. All animals were killed at 90 days and the mesh specimens were subjected to microscopy and immunohistochemistry., Results: Intensely vascularized adhesions to all the implants were observed, although Surgisis showed the lowest percentage of adhesions at each follow-up time. Adhesions had stabilized by 7-14 days. The PTFE meshes were enveloped by a layer of macrophages and connective tissue, bounded by a monolayer of mesothelial cells. Permacol and CollaMend showed similar histological behavior, including cell ingrowth through their fenestrations with no signs of degradation detected at 90 days. In contrast, the Surgisis mesh at 90 days was practically replaced with neoformed tissue., Conclusions: No difference in susceptibility to adhesion formation was noted in the crosslinked collagen meshes compared to PTFE meshes. The noncrosslinked collagen mesh Surgisis showed the best behavior in that it induced fewer adhesions. Ninety days after implant, a more intense macrophage response was observed in CollaMend and Permacol than in PTFE or Surgisis.

Introduction: The intraperitoneal behavior of a prosthetic material used to repair a hernia is key to the success of the postimplant repair process. This study was designed to laparoscopically examine the real-time behavior of three composite meshes incorporating a chemical adhesion barrier when placed in contact with the visceral peritoneum., Material and Methods: The defects of 7 × 5 cm were created in the ventral abdominal wall of 18 New Zealand White rabbits and repaired using Parietex Composite® (n = 6), Sepramesh® (n = 6), or Proceed® (n = 6). At 24 hr, 3, 7, and 14 days postimplant, adhesion formation was quantified by subjecting photographs obtained during laparoscopy to image analysis. At 14 days, specimens of the implants and surrounding host tissue were obtained for histologic, morphometric, and immunohistochemical analyses., Results: There were no cases of infection and/or rejection of the implant. Adhesion formation followed by laparoscopy 3, 7, and 14 days after implant was significantly lower for Parietex® than the other biomaterials. Degradation of the chemical barrier occurred earliest in Sepramesh®, this barrier being most stable at 14 days for the Parietex® implants. Macrophage counts were significantly greater for Sepramesh®. The thickness of the neoformed peritoneum formed on the three implants varied significantly (p < .05): 276.89 ± 38.87 μm, 84.49 ± 19.05 μm, and 161.97 ± 47.05 μm, respectively for Paritex®, Sepramesh®, and Proceed®., Conclusions: (a) The most stable barrier against biodegradation was that of Parietex®; (b) the first postimplant week was the most critical period for adhesion formation; and (c) all three biomaterials showed good intraperitoneal behavior.

Purpose: To evaluate possible changes in the collagen and elastic components of the subepithelial connective tissue of human pterygium., Design: Immunohistochemical study., Methods: Immunohistochemical staining using antitropoelastin, anti-fibulin-2, and anti-fibulin-3 antibodies was performed in 10 normal conjunctival and 20 pterygium specimens. Masson trichome staining also was performed to study subepithelial connective tissue. Sirius red staining was used to identify collagen type I and III components. Tropoelastin, fibulin-2, and fibulin-3 messenger ribonucleic acid (mRNA) expressions were analyzed in 9 conjunctival and 12 pterygium specimens by quantitative real-time polymerase chain reaction assay., Results: The subepithelial connective tissue and vessels were more predominant in pterygium compared with the normal conjunctival tissue. Amorphous subepithelial zones were observed in the areas of the pterygium tissue, but not in normal conjunctiva. Increased tropoelastin staining was seen in the pterygium tissue with areas of degenerative changes or immature formation of elastic fibers, as well an increase in tropoelastin mRNA, in contrast with fibulin-2 and fibulin-3 messenger levels. Fibulin-2 and fibulin-3 expression was colocalized in the subepithelial connective tissue and was distributed along blood and lymphatic vessels. Collagen type III, an immature form of collagen, was increased in the pathologic samples in association with a tissue remodeling process., Conclusions: Elastin metabolism is dysregulated in the pathogenesis of human pterygium with tropoelastin, fibulin-2, and fibulin-3 overexpression in the subepithelial connective tissue., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bombesin (BN) and gastrin-releasing peptide (GRP) have been shown to stimulate the growth of human prostate cancer in vivo and in vitro by mechanisms initiated by binding of the peptide to BN/GRP receptor (GRPR). GRPR is overexpressed in a variety of human cancers, including human prostatic carcinoma. This led us to evaluate the effectiveness of blocking GRPR and of chemotherapy targeted to GRPR in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cells, which exhibit different features of disease progression. Thus, we used a cytotoxic BN/GRP analog, AN-215, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to BN-like carrier peptide, and a BN/GRP receptor antagonist, RC-3095. Semiquantitative RT-PCR and Western blotting revealed that mRNA and protein levels for GRPR increased in prostate cancer cells as compared with nonneoplastic RWPE-1 cells. Immunofluorocytochemistry and Western blot assays revealed that AN-215 was the most effective analog decreasing both the expression of epidermal growth factor receptor family members and the activation of epidermal growth factor receptor and HER-2, which are associated to a poor prognosis. Furthermore, analogs targeted to BN/GRP receptors, AN-215 and RC-3095, blocked the effect of BN on cell growth in RWPE-1, LNCaP and PC-3 cells. These findings shed light on the mechanisms of action of these analogs and support the view that the use of AN-215 and RC-3095 for blocking BN/GRP receptors for targeted therapy may be of benefit for treatment of advanced prostate cancer.

Receptors for vasoactive intestinal peptide (VIP) and the human epidermal growth factor family of tyrosine kinase receptors (HER) are potent promoters of cell proliferation, survival, migration, adhesion and differentiation in prostate cancer cell lines. In this study, we analyzed the cross-talk between both classes of receptors through the regulation of HER2 transactivation and expression by VIP. Three growth-hormone-releasing hormone analogs endowed with antagonistic activity for VIP receptors (JV-1-51, -52, and -53) abrogated the autocrine/paracrine stimuli of VIP on androgen-independent PC3 cells in the absence or the presence of 10% fetal bovine serum. Semiquantitative and real-time quantitative RT-PCR together with Western blotting showed increased expression levels of both mRNA and proteins for HER2 and HER3 in PC3 and androgen-dependent LNCaP prostate cancer cells as compared to non-neoplastic RWPE-1 cells. VIP (100 nM) stimulated the expression levels of both HER2 and HER3 in PC3 cells in a time-dependent manner. Whereas these effects were relatively slow, VIP rapidly (0.5 min) increased HER2 tyrosine phosphorylation. This pattern of HER transactivation was blocked by H89, a protein kinase A (PKA) inhibitor, as well as by the specific VIP antagonist JV-1-53, indicating the involvement of VIP receptors and PKA activity in phosphorylated HER2 formation. These findings support the merit of further studies on the potential usefulness of VIP receptor antagonists and both HER2 antibodies and tyrosine kinase inhibitors for prostate cancer therapy.