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Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-dimensional vascular architecture. We established a robust, high-throughput method to culture endothelial cells as 96 three-dimensional and perfusable microvessels and developed a quantitative, real-time permeability assay to assess their barrier function. Culture conditions were optimized for microvessel formation in 7 days and were viable for over 60 days. The microvessels exhibited a permeability to 20 kDa dextran but not to 150 kDa dextran, which mimics the functionality of vasculature in vivo. Also, a dose-dependent effect of VEGF, TNFα and several cytokines confirmed a physiologically relevant response. The throughput and robustness of this method and assay will allow end-users in vascular biology to make the transition from two-dimensional to three-dimensional culture methods to study vasculature.

Objectives. In this article we describe the long-term follow-up of patients with carcinoma in situ (CIS) of the urinary bladder and examine whether the extent of CIS, the presence of associated papillary tumors, or the response to treatment influence the course of the disease. Methods. Fifty-two patients with CIS of the bladder, treated in a randomized prospec­ tive study, are described. In 23 patients with concomitant papillary tumors all macro- scopically visible lesions were completely resected transurethrally (TUR). CIS was histo­ logically confirmed in all patients by biopsy, 29 of whom had primary CIS. The patients were treated with intravesical mitomycin, bacille Calmette-Guerin (BCG)-RIVM or BCG- Tice and followed regularly by urine cytology, cystoscopy, and biopsy. Results. Complete response was achieved in 65% of the patients. Of these respon­ ders, 24% later had a recurrence of CIS or a superficial tumor and 18% had progres­ sive disease (PD). In the nonresponding patients, progression occurred in 67%. In the whole group, PD was seen in 35% of the patients, and radical cystectomy was per­ formed in 21 %. The disease-related death rate was 13%. The risk for recurrence or PD was not higher in patients with more extensive CIS, defined as three or more positive biopsy results or when CIS was associated with papillary tumors compared to patients with one or two biopsy specimens positive for CIS or CIS alone. Nonresponding patients showed a significantly higher progression rate and cystectomy rate than responding pa­ tients (P = 0.0012 and 0.008, respectively). Conclusions. CIS of the bladder is a malignancy with a poor prognosis, especially in patients not responding after intravesical treatment. Early detection and adjuvant in­ travesical treatment after TUR of concomitant papillary tumors are required. In patients not responding after intravesical treatment, radical surgery is necessary before pro­ gression occurs. The number of biopsies positive for CIS, not the presence of concomi­ tant superficial tumors, was an indicator for progression or recurrence.