Faculty Bibliography

In this report we describe a practical procedure for measuring interstitial fluid pressure (IFP) using fiberoptic pressure transducers based on optical interferometry. Eight mice were used for subcutaneous IFP measurements and four mice for intramuscular IFP measurements with a FOBPS-18 fiberoptic pressure transducer. We used four mice for subcutaneous IFP measurements with a SAMBA-420 MR fiberoptic pressure transducer. One measurement was made for each mouse simultaneously by using a fiberoptic system and an established approach, either transducer-tipped catheter or wick-in-needle technique. The mean IFP values obtained in subcutaneous tissues were -3.00 mm Hg (SEM-/+0.462, n=8), -3.25 mm Hg (SEM-/+0.478, n=4), -3.34 mm Hg (SEM-/+0.312, n=6), and -2.85 (SEM-/+0.57, n=6) for the FOBPS fiberoptic transducer, the SAMBA fiberoptic transducer, the transducer-tipped catheter, and the wick-in-needle technique, respectively. There was no difference between these techniques to measure IFP (Friedman test, p=0.7997). The subcutaneous IFP measurements showed strong linear correlation between fiberoptic transducer and transducer-tipped catheter (R(2)=0.9950) and fiberoptic transducer and wick-in-needle technique (R(2)=0.9966). Fiberoptic pressure transducers measure the interstitial fluid pressure accurately, comparable to conventional techniques. The simplified IFP measurement procedures described in this report will allow investigators to easily measure IFP, and elucidate the unit pressure change per unit volume change (dP/dV) in normal or cancer tissues in the presence of strong electromagnetic fields encountered in MRI.

PURPOSE/OBJECTIVE:The aim of this study is to investigate markers of inflammation and oxidative stress in an early model of diabetic retinopathy, correlate retinal and plasma results and evaluate the influence of treatment by N-acetylcysteine (NAC), a free radical scavenger. METHODS:Four groups were studied: control (C), streptozotocin (STZ)-induced diabetic rats (D), STZ rats following 8 weeks of NAC (DT), and control rats following 8 weeks of NAC (CT). Plasma levels of free 15-F2t-isoprostane (15-F-2t-IsoP), superoxide dismutase (SOD) and tumour necrosis factor-alpha (TNF-alpha) were obtained. Primary antibodies against macrophages (ED-1), microglia (Ox-42), pericytes (NG-2), endothelial and perivascular cells (IB-4), haem oxygenase 1 (HO-1) and vascular endothelial growth factor (VEGF) were used. RESULTS:Expression of NG-2 was robust in C, CT, DT, and mild in D. The intensity of IB-4 was higher in D and DT compared with the C and CT. Ox-42 and ED-1 expression was higher in the D than in the DT, C or CT. Expression of VEGF and HO-1 was non-specific across the four groups. Plasma levels of 15-F-2t-IsoP and TNF-alpha were higher in the D as compared with the C, CT and DT. SOD levels were lower in the D when compared with the C, CT and D. CONCLUSIONS:Macrophage/microglia activation, pericyte loss and endothelial/perivascular cell changes occur early in the pathogenesis of DR. These changes are associated with an increase in plasma markers of oxidative stress and inflammation and are minimized by treatment with NAC. The results suggest that therapies that reduce free radicals will help minimize the early events in diabetic retinopathy in the STZ model.

Research on vascular adaptation to microgravity in the central nervous system requires a simple, noninvasive, direct imaging technique that can be performed with compact equipment. In this report we describe a practical, nonmydriatic, retinal self-imaging technique using a Kowa Genesis-D hand-held digital camera and a Black and Decker laser level. This simple technique will be useful to clinical physiologists conducting microgravity research, as well as for the studies of high-altitude medicine and aviation physiology.

CONTEXT/BACKGROUND:The transgenic human islet amyloid polypeptide (HIP) rat model of type 2 diabetes mellitus (T2DM) parallels the functional and structural changes in human islets with T2DM. OBJECTIVE:The transmission electron microscope (TEM) was utilized to observe the ultrastructural changes in islet microcirculation. METHODS:Pancreatic tissue from male Sprague Dawley rats (2, 4, 8, 14 months) were used as controls (SDC) and compared to the 2-, 4-, 8- and 14-month-old HIP rat models. RESULTS:The 2-month-old HIP model demonstrated no islet or microcirculation remodeling changes when compared to the SDC models. The 4-month-old HIP model demonstrated significant pericapillary amyloid deposition and diminution of pericyte foot processes as compared to the SDC models. The 8-month-old model demonstrated extensive islet amyloid deposition associated with pericyte and beta-cell apoptosis when compared with SDC. The 14-month-old HIP model demonstrated a marked reduction of beta-cells and intra-islet capillaries with near complete replacement of islets by amyloidoses. Increased cellularity in the region of the islet exocrine interface was noted in the 4- to 14-month-old HIP models as compared to SDC. In contrast to intra-islet capillary rarefaction there was noticeable angiogenesis in the islet exocrine interface. Pericytes seemed to be closely associated with collagenosis, intra-islet adipogenesis and angiogenesis in the islet exocrine interface. CONCLUSION/CONCLUSIONS:The above novel findings regarding the microcirculation and pericytes could assist researchers and clinicians in a better morphological understanding of T2DM and lead to new strategies for prevention and treatment of T2DM.

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.

BACKGROUND:The walls of capillaries in prostate cancer are composed of endothelial cells, and pericytes. NG2 is a transmembrane proteoglycan on nascent pericytes with a functional role in neovascularization. METHODS:The anti-angiogenic effect of hydron pellets containing NG2 neutralizing antibody was quantified in intracorneal PC-3 and LNCaP xenografts. TRAMP and TRAMP-C1 tumors grafted in NG2 knockout mice represented intrinsic pericyte targeting. TRAMP and TRAMP-C1 grafts were analyzed with confocal microscope for microvascular density (MVD) and lymphatic vascular density (LVD). RESULTS:NG2 neutralizing antibody decreased corneal neovascularization in PC3 (P<0.0001), and LNCaP (P=0.0079) xenografts. Mean MVD in TRAMP and TRAMP-C1 tumors in NG2 knockout mice were 71% (P=0.0006) and 63% (P=0.0011) lower than wild type controls, respectively. Mean LVD in TRAMP and TRAMP-C1 tumors in NG2 knockout mice were 73% (P=0.0003) and 84% (P<0.0001) lower than wild type controls, respectively. CONCLUSIONS:Targeting of pericyte-NG2 decreases neovascularization and lymphangiogenesis in prostate cancer significantly.

In this investigation, we explored whether knockout of nerve/glial antigen 2 (NG2), a pericyte component, inhibited neovascularization and growth of uveal melanoma xenografts. For this, we used multichannel laser scanning confocal microscopy and quantitative image analysis. Orthotopic human uveal melanoma (OCM-1A) xenografts were induced in NG2 knockout and wild-type mice, which were immunosuppressed with cyclosporin A. Inhibition of pericytes through NG2 proteoglycan decreased neovascularization and tumor end volume, rendering pericytes and NG2 proteoglycan potential cellular and molecular therapeutic targets in uveal melanoma.

PURPOSE/OBJECTIVE:Bone-marrow (BM)-derived hematopoietic precursor cells are thought to participate in the growth of blood vessels during postnatal vasculogenesis. In this investigation, multichannel laser scanning confocal microscopy and quantitative image analysis were used to study the fate of BM-derived hematopoietic precursor cells in corneal neovascularization. METHODS:A BM-reconstituted mouse model was used in which the BM from enhanced green fluorescent protein (GFP)-positive mice was transplanted into C57BL/6 mice. Basic fibroblast growth factor (bFGF) was used to induce corneal neovascularization in mice. The vasculogenic potential of adult BM-derived cells and their progeny were tested in this in vivo model. Seventy-two histologic sections selected by systematic random sampling from four mice were immunostained and imaged with a confocal microscope and analyzed with image-analysis software. RESULTS:BM-derived endothelial cells did not contribute to bFGF-induced neovascularization in the cornea. BM-derived periendothelial vascular mural cells (pericytes) were detected at sites of neovascularization, whereas endothelial cells of blood vessels originated from preexisting blood vessels in limbal capillaries. Fifty three percent of all neovascular pericytes originated from BM, and 47% of them originated from preexisting corneoscleral limbus capillaries. Ninety-six percent and 92% of BM-derived pericytes also expressed CD45 and CD11b, respectively, suggesting their hematopoietic origin from the BM. CONCLUSIONS:Pericytes of new corneal vessels have a dual source: BM and preexisting limbal capillaries. These findings establish BM as a significant effector organ in corneal disorders associated with neovascularization.