Vascularization of solid tumors is thought to occur by sprouting or intussusceptive angiogenesis, co-option of existing vessels, and vasculogenic mimicry after the onset of tumor hypoxia, when the tumor radius exceeds the oxygen diffusion distance. In contrast, here we show that individual endothelial cells that are incorporated into pre-hypoxic tumors give rise to tumor blood vessels via vasculogenesis. Small metastatic lung tumor sections obtained after tail-vein injection of a syngeneic breast cancer cell line in the nude mice were labeled with antibodies against endothelial cell markers. Immunofluorescence showed the incorporation and mixed growth of CD31-, Tie-2-, and CD105-positive endothelial cells in tumors with radii less than oxygen diffusion distance and subsequent development of blood vessels from these early-incorporated endothelial cells. This observation lays the foundation of a novel vasculogenic paradigm of tumor vascularization, where incorporation of endothelial cells and their growth among tumor cells occur before the onset of core hypoxia in lung metastatic tumors.

A rapidly increasing body of data suggests an essential role of endothelial progenitor cells (EPCs) in vascular regeneration, formation of new vessels in cardiovascular diseases and also in tumor vasculogenesis. Moreover, recent data obtained from clinical studies with anti-angiogenic drugs in tumor therapy or with pro-angiogenic stimuli in ischemic disorders implicate a predictive role of the number of EPCs circulating in the peripheral blood in monitoring of these diseases. However, there is still some controversial data regarding the relevance of the EPCs in vascular formation depending on models used and diseases studied. One of the essential prerequisites for a better understanding of the whole contribution of EPCs to vascular formation in adult, a process called postnatal vasculogenesis, is to identify their exact sources. We could recently discover the existence of EPCs in a distinct zone of the vascular wall of large and middle sized adult blood vessels and showed that these cells are capable to differentiate into mature endothelial cells, to form capillary sprouts in arterial ring assay and to build vasa vasorumlike structures within the vascular wall. They also can be mobilized very rapidly from the vascular wall by tumor cells. This review will discuss the functional implications of these vascular wall resident endothelial progenitor cells (VW-EPCs) in relation to those of EPCs circulating in peripheral blood or derived from the bone marrow in cardiovascular and neoplastic diseases.

New blood vessels are formed through angiogenesis and postnatal vasculogenesis. Thus, it is essential to identify vascular stem and progenitor cell niches and the mechanisms governing their role in blood vessel formation. Although much is known about circulating and bone marrow-derived endothelial progenitor cells (EPCs), little is known about the vascular wall as an EPC niche. Experimental evidence strongly suggests that EPCs, as well as other stem and progenitor cells, reside in distinct zones of the vessel wall, such as within the subendothelial space and in the so-called "vasculogenic zone" within the vascular adventitia. In this review, we discuss the potential implications of different types of vascular wall resident stem and progenitor cells in health and disease.

Rete testis and epididymis are rare locations for primary tumors or metastasis. Assuming that this may be related to expression level of angiogenic inhibitors, we focused our study on the expression pattern of collagen 18/endostatin. In situ hybridization and immunohistochemistry for collagen 18 and endostatin were carried out on sections of human rete testis and epididymis as well as on epididymal adenoma and human testicular tissue with or without carcinoma in situ (CIS). In situ hybridization revealed strong expression of collagen 18 mRNA in rete testis, efferent ducts and epididymal duct. Immunostaining showed collagen 18 in epithelium and basement membrane as well as in blood vessels of rete testis. Further, in both efferent ducts and epididymal duct, collagen 18 was mainly localized in the basement membrane of these ducts and of the blood vessel wall. Endostatin immunostaining was localized in the epithelium of rete testis, efferent ducts and epididymal duct. This pattern of endostatin staining was absent in epididymal adenoma tissue while tumor associated blood vessels exhibited strong endostatin staining. No endostatin staining was detectable in normal germinal epithelium and CIS cells while Leydig cells exhibited strong endostatin staining. High endostatin expression in epididymis may protect this organ against tumor development. Gene therapeutic strategies providing high expression of endostatin in normal epithelia may be useful to prevent tumor development.

BACKGROUND: The current TNM bladder cancer staging system stratifies bladder muscle invasion into superficial (pT2a) and deep (pT2b). Controversy exists regarding the significance of the extent of muscle invasion on clinical outcome. OBJECTIVE: Our aim was to compare the cancer-specific outcomes of patients with pT2 urothelial carcinoma of the bladder (UCB) at radical cystectomy (RC) in a large international cohort of patients. DESIGN, SETTING, AND PARTICIPANTS: The records of patients treated with RC for UCB at six centers were reviewed. Of the 2605 reviewed patients, 565 (21.7%) had pT2 disease. None of the patients received preoperative systemic chemotherapy or radiotherapy. MEASUREMENTS: Patients' characteristics and outcome were evaluated. RESULTS AND LIMITATIONS: The median patient age in the entire group was 66.2 yr. Of the 565 patients with pT2 UCB, 249 patients (44.1%) had substage pT2a; 316 patients (55.9%) had pT2b. One hundred and eleven patients (19.6%) had metastases to regional lymph nodes. Median follow-up was 50.5 mo. Recurrence-free survival (73.2% vs 58.7%) and cancer-specific survival (78.0% vs 65.1%) estimates were significantly better for pT2a patients compared with those with pT2b (p=0.002 and p=0.001, respectively). Pathologic T2 substaging was associated with worse recurrence-free and cancer-specific survival after adjusting for the effects of standard pathologic features (p=0.011 and p=0.006, respectively). The statistical significance of these associations was reconfirmed in subgroup analysis limited to those patients with pathologically negative lymph nodes. CONCLUSIONS: In this large international cohort, pathologic substaging helped to stratify patients with lymph node-negative pT2 UCB into statistically significantly different risk groups. These data support the value of the current American Joint Committee on Cancer TNM staging.

PURPOSE: To report on our recent experience with peri- and postoperative morbidity of radical cystectomy in patients 75 years and older compared to younger patients. PATIENTS AND METHODS: Medical records of 326 consecutive patients undergoing radical cystectomy from May 2004 through April 2008 were reviewed. RESULTS: Eighty-five of 326 patients (26%) were >/=75 years (75-95) old. ASA score was equal 3 or greater in 51% of patients >/=75 years and 32% of patients <75 years. Ileal conduit was performed in 83% of patients >/=75, 16% received an ileal neobladder compared to 46 and 51%, respectively, in patients <75. A total of 33 patients (39%) in the older patient group received blood transfusions intraoperatively compared to 76 patients (32%) in the younger age group. In 6 patients >/=75 years (7.1%) and 17 patients <75 (7.1%) open surgical revision was necessary, perioperative complication rate was 22 and 21%, respectively. The most common complications were wound dehiscence (5.9 vs. 7.5%), infections (4.7 vs. 4.6%), and pulmonary embolism (3.5 vs. 2.1%). Perioperative mortality was 1.2%(1 patient) in the elderly versus 0.4%(1 patient) in the younger age group. CONCLUSION: Our data show that radical cystectomy can be offered to the elderly patient with acceptable morbidity. Because of higher comorbidity rate in the elderly, therapeutic decision for radical cystectomy in elderly patients should be made carefully and individually. Nevertheless our results demonstrate that age itself is not a main criterion which has to be considered strongly in decision making for radical cystectomy.

OBJECTIVES: Preoperative Gleason scores (GSs) are often upgraded after pathologic examination of the prostate following radical prostatectomy (RP). There have been disparate reports of the impact of different factors as predictors of GS upgrading after RP. We sought to study the robustness of frequently reported predictors in an unselected single institution cohort. PATIENTS AND METHODS: A total of 684 patients with biopsy-proven prostate cancer treated with RP between 2004 and 2007 were included in the study. The association between clinical and pathologic parameters and GS upgrading was retrospectively evaluated. Logistic regression analysis was used to identify predictors of pathologic grading changes. Likelihood of upgrading was compared between tertile groups for prostate volume and prostate-specific antigen (PSA) density using chi(2) analysis and multivariate logistic regression. Pathologic outcomes were compared between cases with and without GS upgrading. RESULTS: The overall mean age was 64.3 years, with median PSA level of 7.04 ng/ml. Overall, 203 cases (29.7%) were upgraded, whereas 481 patients (70.3%) were downgraded or had identical biopsy and pathologic GS after RP. Patients with prostate volume of <31 g were upgraded in 32.6% of the cases compared with 21.9% in patients with prostate volume of >45 g (P = 0.020). On multivariate analysis preoperative PSA (P < 0.0001), prostate volume (P < 0.0001), and PSA density (P < 0.0001) were predictive of Gleason sum upgrading. Upgraded patients were more likely to have extracapsular extension, seminal vesicle invasion, positive surgical margins, and lymphonodular invasion at RP (P < 0.001, P < 0.001, P < 0.001, and P < 0.001, respectively). CONCLUSIONS: Smaller prostate volume and higher PSA level are associated with clinically significant upgrading of GS. PSA density as a function of both is a significant predictor of GS upgrading in low- and high-risk patients. This may be of relevance in the pretreatment risk assessment of prostate cancer patients.

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 micro vascular 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.

Metastases to regional lymph nodes are a common early event in many malignant diseases and have a poor prognosis, including in urological cancers. Molecular pathways contributing to lymphatic tumour dissemination and lymph node metastasis remain poorly understood. Besides the process of lymphovascular invasion (LVI), recent studies suggested de novo lymphatic vessel formation (i.e. lymphangiogenesis) as a potential mechanism of lymphatic tumour spread. Specific markers for lymphatic endothelium have recently been discovered, enabling basic morphological studies on lymphatic vessel density. There is a gap in the knowledge of the functional relationship between tumoral lymphatic vessels, LVI, lymphangiogenesis and the formation of lymph node metastases. The identification of lymph-specific growth factors (e.g. vascular endothelial growth factor-C and -D) as promoters of lymphatic metastasis has resulted in the interesting idea of targeting the pathways involved in lymphatic tumour progression. We summarize preliminary evidence on the role of lymphangiogenesis during the formation of lymphatic metastasis in the most common urological cancers.

OBJECTIVES: Various imaging modalities, such as magnetic resonance imaging (MRI), have been assessed with regard to their value in the detection of prostate cancer (CaP). However, there is a need for less time-consuming and more cost effective procedures in urology. In order to determine the ability of contrast-enhanced transrectal ultrasound (CE-TRUS) to identify CaP, we investigated patients scheduled for radical prostatectomy for CaP and radical cystoprostatectomy for bladder cancer. MATERIAL AND METHODS: Between May and August 2008, 35 consecutive patients with CaP and muscle-invasive bladder carcinoma were prospectively enrolled in this single center study. All patients underwent B-mode TRUS and CE-TRUS (Sequoia 512 unit with an endocavity probe EV8C4, 8 MHz; Siemens, Erlangen, Germany) by one investigator blinded to any clinical data before radical surgery. Contrast-enhanced images were obtained after intravenous infusion of a bolus (2.4 ml) of the contrast agent SonoVue (Bracco, Milan, Italy). Ultrasound findings (CE-TRUS and B-mode TRUS) were correlated with step-section histology. RESULTS: On a per-patient basis, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for detecting CaP with CE-TRUS were 71.0%, 50.0%, 91.7%, and 18.2%, respectively. In comparison with B-mode TRUS (sensitivity 45.2%, specificity 75.0%, PPV 93.3%, and NPV 18.0%), CE-TRUS performed significantly better (P = 0.004, McNemar test). On a per-prostate-lobe basis sensitivity, specificity, PPV, and NPV were 69.0%, 33.3%, 83.3%, and 18.2%. CONCLUSION: CE-TRUS detected prostate cancer with a modest sensitivity and a high PPV in a selected patient cohort. Future randomized-controlled multicenter studies are needed to further validate the value of CE-TRUS in the detection of CaP. Based on our results, CE-TRUS may not be recommended as a routine procedure in the diagnosis of CaP at present.

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: 207ng/ml vs 0ng/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 110ng/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.

Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

OBJECTIVE: To review the regulating effects of estrogen on endothelial cell functions, the involved endothelial progenitor cells (EPCs), and the related VEGF, and explore the mechanism of estrogen participating in new vessel formation on wound basement and wound healing. METHODS: Recent literature about biological effects of estrogen on capillary vessel formation was reviewed. RESULTS: The formation of new vessel in the wound's granulation tissue contained vasculogenesis and vascularization, and the new vessels could transport the oxygen and nutrient for the metabolism of the local healing tissue. The estrogen effected vascular endothelial cells through its receptors. Peripheral vascular EPCs differentiated into endothelial cells and participated into the formation of new vessels. Estradiol exerted influences on the dynamics of vascular EPCs and the neovascularization. VEGF was a key mediator in the processes of estradiol regulating angiogenesis. CONCLUSION: Understanding the molecular mechanisms that regulate vessel formation in wound healing, especially how estrogen modulates its receptor and angiogenic factor, may provide new approaches for managing wound healing.

Blood vessel formation (neovascularization) in tumors can occur through two mechanisms: angiogenesis and vasculogenesis. Angiogenesis results from proliferation and sprouting of existing blood vessels close to the tumor, while vasculogenesis is believed to arise from recruitment of circulating cells, largely derived from the bone marrow, and de novo clonal formation of blood vessels from these cells. Increasing evidence in animal models indicate that bone marrow-derived endothelial precursor cells (EPC) can contribute to tumor angiogenesis. This review aims to collate existing literature and provide an overview on the current knowledge of EPC involvement in breast cancer angiogenesis. We also discuss recent attempts to use EPC as biomarker and therapeutic target in clinical trials.

Hematopoietic stem cells derive regulatory information also from parathyroid hormone (PTH). To explore the possibility that PTH may have a role in regulation of other stem cells residing in bone marrow, such as mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) we assessed the effect of this hormone on the in vitro behavior of MSCs and EPCs. We evidenced that MSCs were much more responsive to PTH than EPCs. PTH increased the proliferation rate of MSCs with a diminution of senescence and apoptosis. Taken together, our results may suggest a protective effect of PTH on MSCs that reduces stress phenomena and preserve genome integrity. At the opposite, PTH did not modify the fate of EPCs in culture. J. Cell. Physiol.(c) 2009 Wiley-Liss, Inc.

Strong evidence suggests that bone marrow-derived cells play a role in physiological and pathological blood vessel growth in the adult, both by augmenting angiogenesis through the secretion of angiogenic growth factors and by providing a rich source of progenitor cells that can differentiate into mature vascular endothelial cells. This is a true paradigm shift, since adult neovascularization processes were thought to be limited to angiogenesis. The cells that are critical to postnatal blood vessel growth - endothelial progenitor cells - may be analogous to the embryonic angioblast, in that they can circulate, proliferate and participate in the development of vascular networks by differentiating in situ, probably via the formation of cell clusters into mature endothelial cells. Therefore, initial reports have seen analogs to the process of vasculogenesis in the embryo, where the de novo synthesis of vessels occurs through the formation of blood island-like clusters, which subsequently connect and eventually form systemic vasculature. Recent work implicates precursors of endothelial cells in such processes as myocardial ischemia and infarction, limb ischemia, wound healing, atherosclerosis, endogenous endothelial repair and tumor vascularization. These new insights into the vascular biology of endothelial regeneration and repair led to the development of new cell therapeutic strategies to enhance adult neovascularization and re-endothelialization in ischemic cardiovascular diseases.

It is generally accepted that angiogenesis plays a major role in tumor growth and numerous targeting agents directed against angiogenesis pathways have been developed and approved for clinical. In the past years the concept of angiogenesis has developed into a multi-faceted process in which, besides local activation and division of endothelial cells, bone marrow derived progenitor cells (BMDPCs) contribute to neovascularization. A multitude of preclinical and clinical data indicates that the release of BMDPCs influences the response to certain anticancer modalities. In this review we provide an overview of all the preclinical and clinical studies contributing to this hypothesis and translate these findings to the clinic by pointing out to the clinical implications these findings might have. The recent insight in the mechanism of a systemic host response, in response to various treatment modalities have shed new light on the mechanism of tumor regrowth, early recurrence and metastasis formation during or after treatment. This provides various new targets for therapy which can be used to improve conventional chemotherapy. Furthermore it provides a potential explanation why bevacizumab selectively enhances the effectiveness of only certain types of chemotherapy.

Interest in the regulation of blood vessel formation as a mechanism to permit unregulated tumor cell growth was a prescient hypothesis of Dr. Judah Folkman nearly 3 decades ago. Understanding the cellular and molecular mechanisms that effect the recruitment, expansion, and turnover of the tumor microvasculature continues to evolve. While the fundamental paradigms for improving blood flow to growing, injured, diseased, or tumor infiltrated tissues are well known, the potential role of bone marrow derived circulating endothelial progenitor cells (EPCs) to function as postnatal vasculogenic precursors for tumor microvasculature has become a controversial premise. We will briefly review some recently published high profile papers that appear to derive polar interpretations for the role of EPCs in the angiogenic switch and discuss possible reasons for the disparate views in work conducted in both mouse and man.

Throughout development and adult life the vasculature exhibits a remarkably dynamic capacity for growth and repair. The vasculature also plays a pivotal role in the execution of other diverse biologic processes, such as the provisioning of early hematopoietic stem cells during embryonic development or the regulation of vascular tone and blood pressure. Adding to this importance, from an anatomical perspective, the vasculature is clearly an omnipresent organ, with few areas of the body that it does not penetrate. Given these impressive characteristics, it is perhaps to be expected that the vasculature should require, or at least be associated with, a ready supply of stem and progenitor cells. However, somewhat surprisingly, it is only now just beginning to be broadly appreciated that the vasculature plays host to a range of vessel-resident stem and progenitor cells. The possibility that these vessel-resident cells are implicated in processes as diverse as tumor vascularization and adaptive vascular remodeling appears likely, and several exciting avenues for clinical translation are already under investigation. This review explores the various stem and progenitor cell populations that are resident in the microvasculature, endothelium, and vessel walls and vessel-resident cells capable of phenotypic transformation.

Recent studies have found that bone marrow-derived cells give rise to endothelial cells during states of tissue repair and disease. We have found that one key integrin, integrin-alpha4beta1, promotes the homing of circulating endothelial progenitor cells (EPCs) to sites of ongoing tissue repair. This integrin facilitates the adhesion of EPCs to the vascular endothelium in inflamed tissue or within tumors. We demonstrate how to identify, isolate, purify, and characterize EPCs. We also demonstrate in vivo analysis of the roles of bone marrow-derived cells in tumor growth and angiogenesis by demonstrating adoptive transfer, bone marrow transplantation, tumor models, and immunohistochemistry for markers of blood and endothelial vessels. Finally, we show how to characterize cell adhesion mechanisms regulating bone marrow-derived progenitor cell trafficking.