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Biomedical subjects

G A Lutty

Publications and source records attributed to G A Lutty.

13 recordsLinked to original sources
PubMed1992

A new technique for visualization of the human retinal vasculature.

Adenosine diphosphatase, an endothelial cell ectoenzyme that hydrolyses adenosine 5'-diphosphate to adenosine monophosphate, has been used histochemically to study the human retinal vasculature in flat mounts or as flat-embedded tissue that can be sectioned. In all retinas, reaction product was confined almost exclusively to the vasculature, with arteries having more reaction product than veins or capillaries. Adenosine diphosphatase activity was observed in endothelium, smooth-muscle cells, pericytes, and erythrocytes. In the more elaborate version of the technique, the adenosine diphosphatase-incubated retinas were flat embedded in glycol methacrylate. This allowed study of the retinal vasculature in dual perspective: en bloc with the use of dark-field microscopy and specific areas of interest histologically sectioned for further evaluation. The technique is simple and preserves angioarchitecture because the retinas are intact, and patency of the vasculature can be determined.

Adult↗
PubMed1992

Modification of membrane fluidity in melanin-containing cells by low-level microwave radiation.

The treatment of a B16 melanoma cell line with 2.45-GHz pulsed microwaves (10 mW/cm2, 10-microseconds pulses at 100 pps, 1-h exposure; SAR, 0.2 W/kg) resulted in changes of membrane ordering as measured by EPR (electron paramagnetic resonance) reporter techniques. The changes reflected a shift from a more fluid-like phase to a more solid (ordered) state of the cell membrane. Exposure of artificially prepared liposomes that were reconstituted with melanin produced similar results. In contrast, neither B16 melanoma cells treated with 5-Bromo-2-Deoxyuridine (3 micrograms/day x 7 days) to render them amelanotic, nor liposomes prepared without melanin, exhibited the microwave-facilitated increase of ordering. Inhibition of the ordering was achieved by the use of superoxide dismutase (SOD), which strongly implicates oxygen radicals as a cause of the membrane changes. The data indicate that a significant, specific alteration of cell-membrane ordering followed microwave exposure. This alteration was unique to melanotic membranes and was due, at least in part, to the generation of oxygen radicals.

Animals↗
PubMed1992

Increased sensitivity of the non-human primate eye to microwave radiation following ophthalmic drug pretreatment.

Previous studies in our laboratory have established that pulsed microwaves at 2.45 GHz and 10 mW/cm2 are associated with production of corneal endothelial lesions and with disruption of the blood-aqueous barrier in the non-human primate eye. In the study reported here we examined ocular damage in monkeys (M. mulatta and M. fascicularis) following topical treatment with one of two ophthalmic drugs (timolol maleate and pilocarpine) that preceded exposure to pulsed microwaves. Anesthetized monkeys were sham exposed or exposed to pulsed, 2.45 GHz microwaves (10 microseconds, 100 pps) at average power densities of 0.2, 1, 5, 10, or 15 mW/cm2 4 h a day for 3 consecutive days (respective SARs were 0.052, 0.26, 1.3, 2.6, and 3.9 W/kg). Immediately before microwave exposure, one or both eyes were treated topically with one drop of 0.5% timolol maleate or of 2% pilocarpine. Following administration of a drug, we observed a significant reduction in the power-density threshold (from 10 to 1 mW/cm2) for induction of corneal endothelial lesions and for increased vascular permeability of the iris. Diagnostic procedures (in vivo specular microscopy and fluorescein iris angiography) were performed following each exposure protocol. In addition, increased vascular permeability was confirmed with horseradish peroxidase tracer techniques. Although we did not measure intraocular temperatures in experimental animals, the results suggest that a mechanism other than significant heating of the eye is involved. Our data indicate that pulsed microwaves at an average SAR of 0.26 W/kg, if administered after pretreatment with ophthalmic drugs, can produce significant ocular effects in the anesthetized primate.

Administration, Topical↗
PubMed1991

Altered distribution of basic fibroblast growth factor in diabetic retinopathy.

Basic fibroblast growth factor (FGF) is a potent endothelial cell mitogen that has been proposed to play a role in proliferative diabetic retinopathy and other neovascular processes. Our understanding of the in vivo role of basic FGF in the pathogenesis of these disorders is limited. We studied the immunolocalization of basic FGF in 16 clinical cases of diabetic retinopathy to determine whether the normal retinal distribution of basic FGF changed during the development of diabetic retinopathy and correlated with the onset of retinal neovascularization. By using monoclonal and affinity-purified polyclonal antibodies against basic FGF and heparan sulfate proteoglycan (HSPG), we found that basic FGF colocalized with HSPG to vascular basement membranes. As the basement membranes thickened during the progression of diabetic retinopathy, the intraretinal stores of immunoreactive basic FGF and HSPG expanded. With the development of neovascularization, the colocalization of basic FGF and HSPG changed; HSPG localized to basement membranes, while basic FGF localized intracellularly, with only minimal basement membrane immunoreactivity. Incubations of the neovascular fronds with exogenous basic FGF demonstrated multiple HSPG glycosaminoglycan-binding sites for basic FGF, indicating that basic FGF had not been released from the matrix of neovascular fronds by heparitanase digestion.

Antibodies, Monoclonal↗
PubMed1991

Immunolocalization of tissue plasminogen activator in the diabetic and nondiabetic retina and choroid.

Retinal capillary closure is a common finding in many patients with diabetic retinopathy. The cause of this capillary occlusion is unknown. Since occlusions in microthromboembolic disease can occur because of deficiencies in tissue plasminogen activator (tPA) and since systemic tPA decreases with an increasing duration of diabetes mellitus, the immunohistochemical localization of tPA in the retinas and choroids of diabetic and nondiabetic patients was investigated. The localization of tPA was confined to arteries and arterioles in peripheral retinas from nondiabetics. Both veins and arteries were positive in these choroids. Two of three noninsulin-dependent diabetics had normal levels of immunoreactivity in their retinas, and all had normal levels of immunolocalization in their choroids. All but 2 of the 12 insulin-dependent diabetic eyes (IDDM), however, had reduced levels of retinal tPA immunoreactivity which was most pronounced in their peripheral retinas. Seven eyes from patients with IDDM had no reaction product in their peripheral retinas. Two such eyes also had reduced tPA immunoreactivity in their choroidal vessels. Some tPA-positive vessels were observed in the central retinas of these eyes, but the number of positive vessels and amount of reaction product was greatly reduced compared with eyes from nondiabetic patients. These observations suggest that IDDM patients have reduced fibrinolytic activity in their retinas, which might predispose them to thromboembolic disease.

Adult↗
PubMed1989

Localization of basic fibroblast growth factor to the developing capillaries of the bovine retina.

The basic fibroblast growth factor (FGF) is a potent mitogen that has vascular endothelium as one of its principle target cells. Recent work has provided both the complete amino acid sequence of basic FGF and the nucleotide sequence of the genes for both human and bovine basic FGF. Although capillary endothelial cells have been shown to produce basic FGF in vitro and to deposit basic FGF in their extracellular matrix in vitro as well, no direct evidence yet exists for the distribution of basic FGF in vivo. Antipeptide antibodies were prepared against a 15-amino-acid sequence from the amino terminus of basic FGF in order to avoid cross-reactivity with acidic FGF, a protein with 55% overall homology to basic FGF. After affinity purification, these antisera were used to localize the basic fibroblast growth factor in the fetal and adult bovine retina. Immunoreactive material was found in capillaries of the inner nuclear layer, a capillary network undergoing development during the third trimester in the fetal bovine eye. Although the resolution of the technique does not permit a unique assignment of cellular localization, the presence of stain immediately adjacent to the lumen of capillaries suggests that capillary endothelial cells may produce the basic fibroblast growth factor in vivo during vascular development.

Amino Acid Sequence↗
PubMed1988

Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues.

Oxygen free radicals have been demonstrated to be important mediators of postischemic reperfusion injury in a broad variety of tissues; however, the cellular source of free radical generation is still unknown. In this study, electron paramagnetic resonance measurements with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrate that bovine endothelial cells subjected to anoxia and reoxygenation become potent generators of superoxide and hydroxyl free radicals. A prominent DMPO-OH signal aN = aH = 14.9 G is observed on reoxygenation after 45 min of anoxic incubation. Quantitative measurements of this free radical generation and the time course of radical generation are performed. Both superoxide dismutase and catalase totally abolish this radical signal, suggesting that O2 is sequentially reduced from O2-. to H2O2 to OH.. Addition of ethanol resulted in trapping of the ethoxy radical, further confirming the generation of OH.. Endothelial radical generation was shown to cause cell death, as evidenced by trypan blue uptake. Radical generation was partially inhibited and partially scavenged by the xanthine oxidase inhibitor allopurinol. Marked inhibition of radical generation was observed with the potent xanthine oxidase inhibitor oxypurinol. These studies demonstrate that endothelial cells subjected to anoxia and reoxygenation, conditions observed in ischemic and reperfused tissues, generate a burst of superoxide-derived hydroxyl free radicals that in turn cause cell injury and cell death. Most of this free radical generation appears to be from the enzyme xanthine oxidase. Thus, endothelial cell free radical generation may be a central mechanism of cellular injury in postischemic tissues.

Animals↗
PubMed1987

Visualization of a developing vasculature.

The events involved in vasculogenesis still remain obscure. One difficulty has been the techniques employed to visualize angioblasts, i.e., vascular precursors, during the genesis of blood vessels. The retina provides a unique model for studying these events since it is not completely vascularized in some mammals at birth. Using a previously published magnesium-dependent ATPase technique to visualize the developing retinal vasculature and its precursors, and embedding this tissue in JB-4 methacrylate for serial sectioning, has permitted examination of the retinal vasculogenic processes in dual perspective. The technique has permitted observation of the stages in angioblast differentiation and the apparent importance of glycosaminoglycan-rich cell-free spaces in this process. Perhaps the most important observation is that initial vessel formation occurs by coalescence of angioblasts after differentiation in situ.

Adenosine Triphosphatases↗
PubMed1987

Modulation of human lymphocyte proliferation by normal bovine vitreous.

A heat stable antiangiogenic protein (Bovine Vitreal Protein or BVP) isolated from normal bovine vitreous and known to inhibit growth of endothelial and smooth muscle cells was studied for its in vitro effect on human lymphocyte proliferation. Unheated BVP inhibited lymphoproliferation in 12 normal, healthy donors in response to tetanus toxoid (TT) and to Concanavalin A (Con A), but had little effect on responses to irradiated allogeneic peripheral blood mononuclear leucocytes (PBML) or Pokeweed mitogen (PWM). In contrast, heated (95 degrees C/10 min) BVP strongly inhibited (more than 50%) proliferative responses to allogeneic cells, TT, and Con A, and strongly augmented responses to PWM.

Animals↗
PubMed1986

Presence of endothelial cell growth factor activity in normal and diabetic eyes.

Two classes of growth factors affecting endothelial cell proliferation have been found previously in ocular tissues: a heat labile mitogen from retina (RDGF) and a heat stable inhibitor of proliferation from vitreous. The relative amounts of these growth factors in normal and diabetic cadaver eyes were investigated using fetal bovine aortic endothelial cell proliferation as an assay. Equivalent levels of RDGF activity were extracted from diabetic and normal sensory retinas. An extract from pigment epithelium and choroid was found to have similar levels of mitogenic activity, but this activity was not as heat labile as RDGF. Like RDGF, equivalent amounts of mitogen were extracted from diabetic and normal tissue. Normal human vitreous inhibited endothelial cell proliferation, and this activity was enhanced by heating the material (10 min., 95 degrees C). Four of the five individual diabetic vitreous samples of identical postmortem times were mitogenic when not heated, and exhibited little or no inhibitory activity when heated. Vitreous of identical postmortem times was pooled and fractionated by heparin-Sepharose chromatography to determine if the heat labile mitogen in vitreous was RDGF. From the insulin-dependent diabetic (IDDM) pooled vitreous sample, a prominent protein of 18 Kd was eluted from the column with 1.2 M NaC1, a characteristic of RDGF. This work suggests that both RDGF and the vitreous inhibitor are found in human vitreous, but their relative concentrations may change in the diabetic state so that retinal neovascularization from retina can occur.

Animals↗
PubMed1985

Postnatal retinal vascular development of the puppy.

Retinal vascular development during the first three postnatal weeks was studied in 63 purebred beagle puppies. Use of a positive enzyme histochemical reaction for adenosine triphosphatase in the nuclei and nucleoli of vascular cells made visualization of the retinal vasculature possible. Animals were killed by decapitation. Thus, artifacts resulting from use of anesthetics or tracer substances were avoided. In general, this study demonstrates important similarities between canine and human retinal vascular development, and this gives further reason to use of the puppy retina as a superior model for studying retrolental fibroplasia pathogenesis. This staining technique demonstrates undifferentiated cells in the avascular retina that appear to be vascular precursors or angioblasts. Primordial vessels form by organization of differentiating angioblasts that exist in peripheral retinal cystic spaces at birth, or by addition of fully differentiated endothelium; they form unlike neovascularization. Müller cell processes appear to provide a structural matrix throughout the avascular puppy retina on which differentiated angioblasts organize into a vascular network. Arteries develop in beds of primordial capillaries lying near the leading edge of the developing vasculature. This precedes vein formation which occurs through a process involving coalescence of embryonic capillaries which themselves were derived from primordial capillaries. Preliminary examination of eight mongrel kitten retinas prepared by this method clearly indicates that the puppy retina is much more completely vascularized at birth than that of the newborn kitten. Moreover, the rate of postnatal retinal vascularization is significantly faster in the kitten. The kitten vasculature does appear to form by the organization of angioblasts as in the puppy, but kitten angioblasts have a different appearance from those in the puppy.

Adenosine Triphosphatases↗