LOX-catalyzed collagen stabilization is a proximal cause for intrinsic resistance to chemotherapy.

The potential of altering the tumor ECM to improve drug response remains fairly unexplored. To identify targets for modification of the ECM aiming to improve drug response and overcome resistance, we analyzed expression data sets from pre-treatment patient cohorts. Cross-evaluation identified a subset of chemoresistant tumors characterized by increased expression of collagens and collagen-stabilizing enzymes. We demonstrate that strong collagen expression and stabilization sets off a vicious circle of self-propagating hypoxia, malignant signaling, and aberrant angiogenesis that can be broken by an appropriate auxiliary intervention: Interfering with collagen stabilization by inhibition of lysyl oxidases significantly enhanced response to chemotherapy in various tumor models, even in metastatic disease. Inhibition of collagen stabilization by itself can reduce or enhance tumor growth depending on the tumor type. The mechanistical basis for this behavior is the dependence of the individual tumor on nutritional supply on one hand and on high tissue stiffness for FAK signaling on the other.

Synthesis, characterization, and biological properties of composites of hydroxyapatite and hexagonal boron nitride.

Hydroxyapatite (HA), obtained from bovine bones, was successfully reinforced with hexagonal boron nitrite (h-BN). h-BN/HA composites, with BN content up to 1.5 wt %, were sintered at various temperatures between 1000 and 1300°C, in air. Well-sintered samples were obtained after sintering at 1200 and 1300°C. The presence of h-BN contributed to dense, fine, and well-crystallized microstructure. The results of X-ray diffraction analysis and FT-IR spectroscopy showed that the produced composites comprised biphasic ß-TCP/HCA (HCA: carbonate partially substituted HA). High values of mechanical properties were achieved, namely compression strength 155 MPa for the sample 0.5% h-BN/HA and Vickers microhardness of 716 HV for the samples 1.5% h-BN/HA, both sintered at 1300°C. U2OS human bone osteosarcoma proliferation and cell viability showed no adverse effect in the presence of h-BN/HA, suggesting the potential use of the produced materials as safe biomaterials in bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2384-2392, 2018.

3D bioprinting of biomimetic aortic vascular constructs with self-supporting cells.

Cardiovascular diseases are the leading cause of deaths throughout the world. Vascular diseases are mostly treated with autografts and blood vessel transplantations. However, traditional grafting methods have several problems including lack of suitable harvest sites, additional surgical costs for harvesting procedure, pain, infection, lack of donors, and even no substitutes at all. Recently, tissue engineering and regenerative medicine approaches are used to regenerate damaged or diseased tissues. Most of the tissue engineering investigations have been based on the cell seeding into scaffolds by providing a suitable environment for cell attachment, proliferation, and differentiation. Because of the challenges such as difficulties in seeding cells spatially, rejection, and inflammation of biomaterials used, the recent tissue engineering studies focus on scaffold-free techniques. In this paper, the development of novel computer aided algorithms and methods are developed for 3D bioprinting of scaffold-free biomimetic macrovascular structures. Computer model mimicking a real human aorta is generated using imaging techniques and the proposed computational algorithms. An optimized three-dimensional bioprinting path planning are developed with the proposed self-supported model. Mouse embryonic fibroblast (MEF) cell aggregates and support structures (hydrogels) are 3D bioprinted layer-by-layer according to the proposed self-supported method to form an aortic tissue construct.

Early expression of pregnancy-specific glycoprotein 22 (PSG22) by trophoblast cells modulates angiogenesis in mice.

Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on Gestation Day 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells, and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and PSG23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions that include their ability to induce anti-inflammatory cytokines and proangiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.

Human pregnancy specific beta-1-glycoprotein 1 (PSG1) has a potential role in placental vascular morphogenesis.

Previous studies suggest that human pregnancy specific beta-1-glycoproteins (PSGs) play immunomodulatory roles during pregnancy; however, other possible functions of PSGs have yet to be explored. We have observed that PSGs induce transforming growth factor beta 1 (TGFB1), which among its other diverse functions inhibits T-cell function and has proangiogenic properties. The present study investigates a potential role for PSG1, the most abundant PSG in maternal serum, as a possible inducer of proangiogenic growth factors known to play an important role in establishment of the vasculature at the maternal-fetal interface. To this end, we measured TGFB1, vascular endothelial growth factors (VEGFs) A and C, and placental growth factor (PGF) protein levels in several cell types after PSG1 treatment. In addition, tube formation and wound healing assays were performed to investigate a possible direct interaction between PSG1 and endothelial cells. PSG1 induced up-regulation of both TGFB1 and VEGFA in human monocytes, macrophages, and two human extravillous trophoblast cell lines. We did not observe induction of VEGFC or PGF by PSG1 in any of the cells tested. PSG1 treatment resulted in endothelial tube formation in the presence and absence of VEGFA. Site-directed mutagenesis was performed to map the essential regions within the N-domain of PSG1 required for functional activity. We found that the aspartic acid at position 95, previously believed to be required for binding of PSGs to cells, is not required for PSG1 activity but that the amino acids implicated in the formation of a salt bridge within the N-domain are essential for PSG1 function.

CEACAM1: a novel urinary marker for bladder cancer detection.

BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein; CEACAM1) is expressed in normal bladder urothelium and in angiogenically activated endothelial cells, where it exhibits proangiogenic properties. OBJECTIVE: The aim of this study was to evaluate the value of urinary CEACAM1 for detection of urothelial carcinoma of the bladder (UCB). DESIGN, SETTING, AND PARTICIPANTS: This prospective study included 175 patients. MEASUREMENTS: Immunohistochemistry for CEACAM1 was performed on UCB sections of 10 patients. Enzyme-linked immunosorbent assay (ELISA) for CEACAM1 was performed on urine specimens of healthy volunteers (n=30), patients with benign prostatic hyperplasia (BPH; n=5), severe cystitis (n=5), non-muscle-invasive UCB (n=72), muscle-invasive UCB (n=21), or past history of UCB without evidence of disease (n=42). Western blot analysis was performed on a subgroup of these subjects (n=53). RESULTS AND LIMITATIONS: CEACAM1 immunostaining in normal urothelium disappears in noninvasive UCB but appears in endothelial cells of adjacent vessels. Western blotting revealed presence of CEACAM1 in the urine of no healthy volunteers, of 76% of noninvasive UCB patients, and of 100% of invasive UCB patients. ELISA analysis confirmed that urinary CEACAM1 levels were significantly higher in UCB patients compared with control subjects (median: 207 ng/ml vs 0 ng/ml; p<0.001). The area under the curve for UCB detection was 0.870 (95% confidence interval [CI]: 0.810-0.931). In multivariable logistic regression analyses that adjusted for the effects of age and gender, higher CEACAM1 levels were associated with cancer presence (hazard ratio [HR]: 2.89; 95% CI: 2.01-4.15; p<0.001) and muscle-invasive cancer (HR: 5.53; 95% CI: 1.68-18.24; p=0.005). The cut-off level of 110 ng/ml yielded sensitivity of 74% and specificity of 95% for detecting UCB. Sensitivity was 88% for detecting high-grade UCB and 100% for detecting invasive-stage UCB. Larger studies are necessary to establish the diagnostic and prognostic roles of this highly promising novel marker in UCB. CONCLUSIONS: Urinary CEACAM1 levels discriminate UCB patients from non-UCB subjects. Moreover, urinary levels of CEACAM1 increased with advancing stage and grade.

Pro-angiogenic properties of orosomucoid (ORM).

The acute phase protein orosomucoid (ORM), also known as alpha1-acid glycoprotein (AGP), is found to be increased in infection, inflammation and cancer. Recently, we demonstrated that ORM is produced by endothelial cells and detectable in urine samples of patients with bladder cancer. However, it was not clarified yet whether ORM plays a role in new vessel formation. To this aim we performed overexpression and gene silencing for ORM in human microvascular endothelial cells (HDMECs). ORM purified from human plasma was used individually or in combination with VEGF-A in endothelial tube formation, migration and proliferation assay. The in vivo effect of ORM in angiogenesis was studied using the chicken chorionallantois membrane (CAM) with subsequent counting of blood vessels on histological sections from the stimulated areas of CAM tissue. Our data show that ORM alone enhances migration but not proliferation of HDMECs. ORM alone does not induce endothelial tubes in vitro but simultaneous application of ORM with VEGF-A increases the number and the network of VEGF-A-induced endothelial tubes. Remarkably, ORM alone induces new vessel formation in vivo using CAM assay and supports the VEGF-A-induced new vessel formation in this assay. Taken together, our results let assume that ORM has pro-angiogenic properties and supports the angiogenic effect of VEGF-A. Thus, ORM seems to be involved in the regulation of angiogenesis.

Molecular imaging of tumor blood vessels in prostate cancer.

In the past three decades many efforts have been undertaken to understand the mechanisms of tumor angiogenesis. The introduction of anti-angiogenic drugs in tumor therapy during the last few years necessitates the establishment of new techniques enabling molecular imaging of tumor vascular remodelling. The determination of tumor size as commonly used is not appropriate since the extended necrosis under anti-angiogenic therapy does not necessarily result in the reduction of tumor diameter. The basis for the molecular imaging of tumor blood vessels is the remodelling of the tumor vessels under anti-angiogenic therapy which obviously occurs at an early stage and seems to be a convincing parameter. Beside the enormous progress in this field during the last few years the resolution is still not high enough to evaluate the remodelling of the micro tumor vessels. New imaging approaches combining specific molecular markers for tumor vessels with the different imaging techniques are needed to overcome this issue as exemplarily discussed for prostate cancer in this review. Molecular contrast agents targeting the vasculature will allow clinicians the visualization of vascular remodelling processes taking place under anti-angiogenic therapy and improve tumor diagnosis and follow-up.

Lymphatic reprogramming of microvascular endothelial cells by CEA-related cell adhesion molecule-1 via interaction with VEGFR-3 and Prox1.

Here, we demonstrate that carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed and co-localized with podoplanin in lymphatic endothelial cells (LECs) of tumor but not of normal tissue. CEACAM1 overexpression in human dermal microvascular endothelial cells (HDMECs) results in a significant increase of podoplanin-positive cells in fluorescence-activated cell sorting analyses, while such effects are not observed in CEACAM1 overexpressing human umbilical vein endothelial cell (HUVECs). This effect of CEACAM1 is ceased when HDMECs are transfected with CEACAM1/y- missing the tyrosine residues in its cytoplasmic domain. CEACAM1 overexpression in HDMECs leads to an up-regulation of vascular endothelial growth factor C, -D (VEGF-C, -D) and their receptor vascular endothelial growth factor receptor 3 (VEGFR-3) at mRNA and protein levels. HDMECs transfected with CEACAM1 but not those with CEACAM1/y- show enhanced expression of the lymphatic markers Prox1, podoplanin, and LYVE-1. Furthermore, Prox1 silencing in HDMECs via small interfering RNA blocks the CEACAM1-induced increase of VEGFR-3 expression. Number and network of endothelial tubes induced by VEGF-C and -D are enhanced in CEACAM1-overexpressing HDMECs. Moreover, VEGF-A treatment of CEACAM1-silenced HDMECs restores their survival but not that with VEGF-C and VEGF-D. These data imply that the interaction of CEACAM1 with Prox1 and VEGFR-3 plays a crucial role in tumor lymphangiogenesis and reprogramming of vascular endothelial cells to LECs. CEACAM1-induced signaling effects appear to be dependent on the presence of tyrosine residues in the CEACAM1 cytoplasmic domain.

Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer.

Identification and characterization of biomarkers in body fluids such as serum or urine serve as a basis for early detection of diseases, particularly of cancer. Performing 2-DE with subsequent MS analyses, conventional immunoblotting and immunohistochemistry we identified two proteins, orosomucoid (ORM) and human zinc-alpha(2)-glycoprotein (ZAG), which were increased in the urine samples of patients with bladder cancer in comparison to the urine samples of healthy volunteers. The highest amount of both proteins was found in invasive bladder cancer stages such as pT2-3. Immunohistochemical studies showed ORM in inflammatory cells but also in endothelial cells of blood vessels within or adjacent to the tumor area and in part of the tumor cells. ZAG was prominent in tumor cells at the tumor invasion front. Additionally, ZAG was localized at the luminal surface of normal urothelium, which switches to the basal side when a superficial papillary tumor was observed. These results show that we have been able to identify two new proteins that may be related to the development of superficial bladder cancer and to its switch to an invasive phenotype.

Dual role of carcinoembryonic antigen-related cell adhesion molecule 1 in angiogenesis and invasion of human urinary bladder cancer.

Here, we show that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in umbrella cells of bladder urothelium but is down-regulated in superficial bladder cancer, such as histologic tumor stage a (pTa) and transitional cell carcinoma in situ (pTis). Concurrently, CEACAM1 is up-regulated in the endothelia of adjacent angiogenic blood vessels. Mimicking the CEACAM1 down-regulation in the urothelium, CEACAM1 was silenced in bladder cancer cell lines 486p and RT4 using the small interfering RNA technique. CEACAM1 down-regulation was confirmed at the protein level by Western blot analyses. CEACAM1 silencing leads to a significant up-regulation of vascular endothelial growth factor (VEGF)-C and VEGF-D in quantitative reverse transcription-PCR. Correspondingly, supernatants from the CEACAM1-overexpressing bladder cancer cell lines reduce, but those from CEACAM1 silencing induce endothelial tube formation and potentiate the morphogenetic effects of VEGF. These data suggest that the epithelial down-regulation of CEACAM1 induces angiogenesis via increased expression of VEGF-C and VEGF-D. Inversely, CEACAM1 is up-regulated in endothelial cells of angiogenic blood vessels. This in turn is involved in the switch from noninvasive and nonvascularized to invasive and vascularized bladder cancer. CEACAM1 appears to be a promising endothelial target for bladder cancer therapy.

Design and evaluation of a mucoadhesive therapeutic agent delivery system for postoperative chemotherapy in superficial bladder cancer.

The most common treatment method is known as the transurethral resection (TUR) for the therapy of bladder cancer. Unfortunately, because of the recurrency of the tumoral tissues after TUR the chemotherapy or immunotherapy should be performed. In these kind of therapies the pharmacotherapeutics are infused intravesically into the bladder after TUR periodically (i.e. upto 6--36 weeks, each week). But these therapies are having very big problems (i.e. disturbancy to patients, adjustment of the suitable dosage, loss of active agents without using etc.). An alternative approach can be proposed as to design a pharmacotherapeutic agent delivery system, which will supply the suitable dosage of the agent for a certain time period to solve those problems. In this study; the pharmacotherapeutic agent (i.e. Mytomycin-C) delivery system was prepared by using a mucoadhesive polymer (i.e. chitosan) in the form of cylindirical geometry to facilitate the insertion of the carrier for in vivo studies. The chitosan carriers were prepared by cross-linking during the solvent evaporation technique. In the preparation of the chitosan carriers the chitosan polymers with different molecular weights, different amounts of cross-linker (i.e. glutaraldehyde) and different amounts of pharmacotherapeutic agent were used to obtain desired attachment onto the bladder wall and optimum release rate of the agent. On the other hand because of the gelous structure of the chitosan, the swelling behaviour of the polymer was evaluated by gravimetric determinations in aqueous media periodically.