Faculty Bibliography

A novel procedure using a polyurethane transducer-tipped catheter (Millar) is described that allows reliable measurement of interstitial fluid pressure (IFP) in cancer tissues. Before and after each use, the transducer is calibrated at 37 degrees C by a water column. After calibration, the transducer is passed through the lumen of a surgical needle. The sensor is kept in the lumen of the needle during penetration into the tumor. The sensor tip is then introduced into the center core of the tumor as the needle sleeve is withdrawn from the tumor surface. Our new technique is simple and provides IFPs equal to those provided by the well-established, wick-in-needle technique. Using our new technique, we compared IFP in skin melanoma grafts in NG2 knockout and wild-type mice. Knocking out NG2 proteoglycan on vasculogenic and angiogenic pericytes reduced interstitial fluid pressure in melanoma from +4.9 cm H2O to -0.4 cm H2O (P=0.0054 Mann-Whitney U test).

Bone marrow (BM)-derived cells are thought to participate in the growth of blood vessels during postnatal vascular regeneration and tumor growth, a process previously attributed to stem and precursor cells differentiating to endothelial cells. We used multichannel laser scanning confocal microscopy of whole-mounted tissues to study angiogenesis in chimeric mice created by reconstituting C57BL mice with genetically marked syngeneic BM. We show that BM-derived endothelial cells do not significantly contribute to tumor- or cytokine-induced neoangiogenesis. Instead, BM-derived periendothelial vascular mural cells were persistently detected at sites of tumor- or vascular endothelial growth factor-induced angiogenesis. Subpopulations of these cells expressed the pericyte-specific NG2 proteoglycan, or the hematopoietic markers CD11b and CD45, but did not detectably express the smooth muscle markers smooth muscle alpha-actin or desmin. Thus, the major contribution of the BM to angiogenic processes is not endothelial, but may come from progenitors for periendothelial vascular mural and hematopoietic effector cells.

The NG2 proteoglycan is expressed by nascent pericytes during the early stages of angiogenesis. To investigate the functional role of NG2 in neovascularization, we have compared pathological retinal and corneal angiogenesis in wild type and NG2 null mice. During ischemic retinal neovascularization, ectopic vessels protruding into the vitreous occur twice as frequently in wild type retinas as in NG2 null retinas. In the NG2 knock-out retina, proliferation of both pericytes and endothelial cells is significantly reduced, and the pericyte:endothelial cell ratio falls to 0.24 from the wild type value of 0.86. Similarly, bFGF-induced angiogenesis is reduced more than four-fold in the NG2 null cornea compared to that seen in the wild type retina. Significantly, NG2 antibody is effective in reducing angiogenesis in the wild type cornea, suggesting that the proteoglycan can be an effective target for anti-angiogenic therapy. These experiments therefore demonstrate both the functional importance of NG2 in pericyte development and the feasibility of using pericytes as anti-angiogenic targets.

Apart from tumor-driven neovascularization, a less-appreciated consequence of neurofibromatosis type 1 (NF1) is the hyperproliferation of vascular mural cells (pericytes). This study aims at establishing a role for pericytes in NF1, and determining whether interference with the function of a key pericyte component (NG2 proteoglycan) inhibits NF1 tumor neovascularization. Neovascularization in NF1 was studied in Nf+/+(control), Nf1+/-, and Nf1-/-embryos at E-10, ischemia-induced retinal angiogenesis model in 24 eyes of Nf1+/-, Nf1+/+mice, and in malignant peripheral nerve sheath tumors (MPNSTs) derived from NF1 patients (ST88-14, NMS-2PC) orthotopically grown in nude mice (Crl: nu/nu). The anti-angiogenic effect of intracorneal polymer pellets containing anti-NG2 neutralizing antibody was quantified in the nude-mouse corneal angiogenesis model in which angiogenesis was induced by xenografting NMS-2PC tumor into the corneal stroma of 22 eyes. By using confocal microscopy, immunohistochemistry, and BrdU proliferation assay, the pericyte/endothelium ratios and proliferation rates were measured. Activated pericytes were present at the leading tip of the angiogenic sprouts. Pericytes showed continuous investment of endothelium in both NMS-2PC and ST88-14 MPNST tumor xenografts. Mean corneal angiogenesis induced by NMS-2PC tumor grafts in NG2-antibody treated eyes was 1.491 and 3.186 mm2 in isotype-matched non-immunoglobulin treated eyes (control) (P=0.0002). A total of 193.8 vascular nuclei (a measure of ischemia-induced retinal angiogenesis) was present in angiogenic retinal tufts in Nf1+/- mice compared to 89.23 in Nf1+/+ mice (control) (P<0.0001). Mean pericyte/endothelium investment ratios were 1.015, 1.380, and 2.084 in control, Nf1+/-, and Nf1-/-embryos, respectively. Pericytes were 23% (control), 49% (Nf1+/-), and 69% (Nf1-/-) BrdU-positive. Endothelial cells from the same embryos were 29% (control), 47% (Nf1+/-), and 62% (Nf1-/-) BrdU-positive. Angiogenesis is accelerated in NF1 due to hyperproliferation of pericytes and endothelial cells. Mitotically activated NG2-positive pericytes, and endothelial cells may serve as potential therapeutic targets in NF1.

PURPOSE/OBJECTIVE:To determine the association between anticytomegalovirus (CMV) maintenance therapy after immune recovery and immune recovery uveitis in acquired immunodeficiency syndrome (AIDS) patients on highly active antiretroviral therapy (HAART). DESIGN/METHODS:Observational cohort study. METHODS:Data were obtained on AIDS patients with CMV retinitis followed up at the AIDS Ocular Research Unit of University of California San Diego from November 1995 to October 1999. Immune recovery was defined as CD4 count greater than 50 cells/microl for more than 3 months. Patients with immune recovery uveitis presented with vitritis, cystoid macular edema, or epiretinal membrane. Statistical analyses were conducted to determine the risk of continued use of anti-CMV therapy after immune recovery and the relationship of developing immune recovery uveitis with the type of anti-CMV therapy. RESULTS:Forty-three patients (64 eyes) had healed CMV retinitis and had achieved immune recovery. Thirty-one patients (48 eyes) received anti-CMV therapy after immune recovery, and 20 patients (29 eyes) developed immune recovery uveitis. Per-eye analyses revealed a 3.8-fold increase in the odds of developing immune recovery uveitis with anti-CMV therapy compared with no treatment (P =.02). If treated with cidofovir the odds were 3.3 greater than if treated with an alternative regimen (P =.04), 4.1 greater if treated intravenously (P =.01), and 5.2 greater than if not treated (P =.004). If not treated with cidofovir, a nonsignificant increase in the risk (2.4) of immune recovery uveitis was found (P =.15). Neither the potency nor the use of implants for noncidofovir treatment was related to the risk of recovery uveitis (P >.62). CONCLUSIONS:The use of cidofovir is a primary risk factor in the subsequent development of immune recovery uveitis. Ongoing treatment of healed CMV retinitis after immune recovery does not appear to protect against the development of immune recovery uveitis.

Immunostaining with endothelial and pericyte markers was used to evaluate the cellular composition of angiogenic sprouts in several types of tumors and in the developing retina. Confocal microscopy revealed that, in addition to conventional endothelial tubes heavily invested by pericytes, all tissues contained small populations of endothelium-free pericyte tubes in which nerve/glial antigen 2 (NG2) positive, platelet-derived growth factor beta (PDGF beta ) receptor-positive perivascular cells formed the lumen of the microvessel. Perfusion of tumor-bearing mice with FITC-dextran, followed by immunohistochemical staining of tumor vasculature, demonstrated direct apposition of pericytes to FITC-dextran in the lumen, confirming functional connection of the pericyte tube to the circulation. Transplantation of prostate and mammary tumor fragments into NG2-null mice led to the formation of tumor microvasculature that was invariably NG2-negative, demonstrating that pericytes associated with tumor microvessels are derived from the host rather than from the conversion of tumor cells to a pericyte phenotype. The existence of pericyte tubes reflects the early participation of pericytes in the process of angiogenic sprouting. The ability to study these precocious contributions of pericytes to neovascularization depends heavily on the use of NG2 and PDGF beta -receptor as reliable early markers for activated pericytes.

PURPOSE/OBJECTIVE:We studied the effects of intravitreally administered prinomastat on the take rate and growth of uveal melanoma after xenograft implantation in rabbit uveal melanoma model. METHODS:Uveal melanoma xenograft was implanted to suprachoroidal space in each eye of 24 pigmented rabbits which were immunosuppressed with cyclosporine. One week after surgery, the eyes were randomized to receive prinomastat or the vehicle of the prinomastat intravitreally every week for 4 weeks. The take rate of the xenograft, tumor height, apoptosis, and necrosis in the eyes which developed tumors from the treatment and control groups were compared. RESULTS:A tumor mass was identified in 8 of 24 (33%) prinomastat-treated eyes and 20 of 24 (83%) of the vehicle-treated eyes. Echographic measurements revealed a mean tumor height of 2.2 mm in the prinomastat-treated group and 3.8 mm in the control group in those eyes with take of tumor (p < 0.001). Stereomicroscopic measurements showed a mean tumor height of 1.9 mm in the treatment group and 3.9 mm in the control group (p < 0.001). The mean number of apoptotic nuclei detected per mm(2) of the histologic section in the non-necrotic tumor was 8.12 in the prinomastat-treated group and 0.57 in the control group (p < 0.001). Evaluation of the digital images in microscopic sections of the tumors on histologic slides revealed 29.6% necrosis in prinomastat-treated eyes as compared to 10.9% in vehicle-treated eyes (p = 0.003). CONCLUSIONS:These results suggest that prinomastat treatment significantly reduces the take rate and the growth rate of xenograft in uveal melanoma rabbit model.