Pericyte coverage of retinal and cerebral capillaries

We performed electron microscopic morphometric analyses on capillaries from macular and peripheral retinas of five adult cynomolgous monkeys and three elderly human subjects. Measurements from the monkey retinal capillaries were compared to those made on capillaries from frontal, temporal, parietal, and occipital cerebral cortex of the same animals. We measured the percent coverage of the endothelial lining of the capillaries by pericyte processes, as well as the ratio of the cytoplasmic areas of pericytes and endothelial cells. In addition, we compared the thickness of the capillary basement membranes in three regions: overlying pericytes; overlying endothelial cells; and interposed between pericytes and endothelial cells. In both monkey and human retinas, pericyte processes covered greater than 85% of the circumference of the capillary endothelial tube, whereas pericyte coverage of monkey cerebral capillaries was highly significantly (P less than 0.001) less than that of capillaries in either the macular or peripheral retina. The ratio of pericyte to endothelial cell cytoplasmic areas also was lower in the monkey cerebral cortex than in the retina, though the statistical significance was less than that of the length measurements. In all tissues measured, both from monkeys and humans, the portions of the capillary basement membranes interposed between pericytes and endothelial cells were highly significantly (P less than 0.0001) thinner than the regions of capillary basement membranes covering pericytes and endothelial cells. Considering functions that have been proposed for pericytes, these measurements suggest that regional control of microcirculatory flow and of blood-tissue barrier integrity, as well as control of endothelial cell proliferation, should be much greater in the retina than in the cerebral cortex. Thinner basement membranes between pericytes and endothelial cells may permit more cell membrane contacts between these cells, thus facilitating such control.     

Impact of short-term versus long-term topical steroids on corneal neovascularization after non-high-risk keratoplasty

PURPOSE: To analyze incidence and extent of corneal neovascularization (CN) after non-high-risk keratoplasty and to find out whether duration of postoperative topical steroid therapy (6 vs 12 months) affects CN, corneal endothelial cell count, pachymetry, aqueous flare values, and best-corrected visual acuity at 1 year after keratoplasty. METHODS: Patients of the prospective Erlangen non-high-risk keratoplasty study with available high-quality corneal photographs taken preoperatively and 1 year later were analyzed (n=136). Corneal photographs were evaluated by two independent observers in a standardized semiquantitative fashion. Slides were projected with 100x magnification and corneal vessels classified into five grades with regard to the limbus, sutures and host-graft junction in each of 12 corneal sectors. Incidence and extent of CN after keratoplasty and relation to short-term (0-6 months) versus long-term (0-12 months) postoperative topical steroid therapy were analyzed. The effect of duration of topical steroid therapy on corneal endothelial cell count, pachymetry, aqueous flare values, and best corrected visual acuity was also analyzed. Of the 136 patients, 69 (51%) were randomly assigned to short-term and 67 to long-term topical prednisolone acetate 1%. RESULTS: Fifty-eight percent of patients (n=79) developed a CN within 1 year after keratoplasty in at least one corneal sector (mean 3.1 +/- 2.2, range 1-10). At 1 year after keratoplasty, only in 12% of these patients did at least one vessel reach the host-graft junction or grow into the donor cornea, whereas in 51% vessels were seen beyond the outer suture ends of the double running suture without reaching the host-graft junction. In 37%, capillaries were located between limbus and outer suture ends. New vessels usually pointed directly or indirectly to the outer suture ends and usually were located around the 12 o'clock and 6 o'clock positions. There was no significant difference regarding incidence and extent of CN 1 year after keratoplasty between the long-term and the short-term group. Duration of topical steroid therapy had no significant effect on corneal endothelial cell count and thickness, aqueous flare values and best-corrected visual acuity at 6 and 12 months postoperatively (only at 12 months, corneas in the long-term treatment group were slightly thicker; P=0.03). Interobserver correlation of vessel assessment was 0.77 (Kendall's tau B). CONCLUSIONS: CN is a common phenomenon after non-high-risk keratoplasty. New vessels rarely reach the host-graft junction, most commonly develop from the 6 o'clock and 12 o'clock positions and are usually located between epithelium and Bowman's layer (i.e., at the level of the superficial suture). The direction of vessel growth from the limbus towards the outer suture ends suggests release of angiogenic factors in this area. Prolongation of topical steroid therapy after non-high-risk keratoplasty beyond 6 months in this study did not significantly influence incidence and extent of CN, corneal endothelial cell count, aqueous flare values and best-corrected visual acuity observed 1 year after keratoplasty.     

Pericyte form and distribution in rat retinal and uveal capillaries

Ultrastructural morphometric techniques were used to assess differences in endothelial cells and in pericyte structure and distribution in rat retinal and uveal capillaries. Retinal capillaries were significantly smaller than those in the three different uveal vascular beds, all of which were similar in size. Approximately 10% of the capillaries in the retina and choroid were formed by three endothelial cells, compared with 30% and 46% of capillaries sampled from ciliary processes and iris, respectively. The percentage of the capillary circumference covered by pericytes (46-58%) and the percentage of capillary sections with pericyte nuclei (12-16%) were similar in retina, iris, and ciliary processes. Corresponding data for the choriocapillaris indicated that pericyte coverage of these capillaries was approximately 50% of that observed in the other eye microcirculations. The number of pericyte processes per capillary varied markedly in the different vasculatures, with an average of three for capillaries in the retina and choriocapillaris and nine to eleven for capillaries in the iris and ciliary processes. These marked differences in capillary dimensions are consistent with the well-known capillary hemodynamic and functional differences of these tissues; however, the significance of the differences in pericyte shape, frequency and distribution in the different vasculatures of the eye is less clear.     

Pericyte changes in branch retinal vein occlusion

The model of experimental branch vein occlusion (BVO) in the monkey offers the opportunity to examine retinal capillaries under stress. Electron microscopic morphometry was done on 812 capillaries of 13 eyes of cynomolgus monkeys, comparing 579 capillary collaterals of 9 BVO eyes with 233 normal capillaries of 4 control eyes. The tissue underwent the myosin subfragment-1 technique to decorate and quantify bundles of actin filaments in capillary pericytes. The duration of BVO was 2-48 months. Capillary collaterals of BVO eyes had an enlarged caliber, endothelial hyperplasia, and pericyte hypertrophy, but no proportional increase in basement membrane area. Collaterals near the inner plexiform layer (IPL) had a greater wall thickness, pericyte coverage, and actin coverage than collaterals near the outer plexiform layer (OPL). Pericyte hypertrophy was proportionate to caliber increase in OPL vessels and exceeded caliber increase only in IPL vessels. Actin coverage was proportional with the vessel dilation and size of pericyte cytoplasm in all vessels. These findings indicate that capillary collaterals in BVO are not equipped morphologically for an increased regulatory role in microvascular flow beyond their normal function.     

Effect of aldose reductase inhibitors on the progression of retinopathy in galactose-fed dogs

The onset and progression of diabetic-like retinopathy has been investigated in age-matched male beagle dogs fed a 30% galactose diet. Examination of the intact retinal vessels, isolated by gentle trypsin digestion, reveals that the destruction of pericytes to form pericyte ghosts is the earliest observable retinal vessel change. This results in an irregular distribution of endothelial cell nuclei near pericyte ghosts and the subsequent formation of acellular capillaries containing neither endothelial cells or pericytes. This was followed by the histological appearance of microaneurysms and later, by the funduscopic appearance of intraretinal hemorrhages. Studies in similar galactose-fed dogs treated with aldose reductase inhibitors indicate that all of these changes are linked to the initial aldose reductase associated destruction of pericytes.     

Contribution of bone marrow-derived pericyte precursor cells to corneal vasculogenesis

PURPOSE: 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.     

Absence of degenerative changes in retinal and uveal capillary pericytes in diabetic rats

Ultrastructural morphometric techniques were used to quantify pericyte degeneration in retinal and uveal capillaries of streptozotocin-diabetic rats in order to assess the suitability of this small rodent model of diabetes for studies of the pathogenesis of microvascular eye disease in diabetic humans. Male, Sprague-Dawley rats were killed by intraaortic perfusion of fixative 6 and 9 mos after induction of diabetes with 50 mg/kg streptozotocin. No differences were evident between diabetics and age-matched controls in capillary circumference, numbers of endothelial cells per capillary, and capillary cytoplasmic area of retinal, choroidal, and iridial vessels. Capillary basement membrane width and the percentage of the capillary circumference covered by pericytes were increased in retinas of diabetic vs age-matched control rats after 9 mos of diabetes (P less than 0.05), but no differences were evident in the number of pericyte processes per capillary and the percentage of vessels with pericyte nuclei. No differences in pericyte distributions were observed between control and diabetic rats in the choriocapillaris and iris after 9 mos of diabetes. These findings indicate that retinal capillary basement membrane thickening precedes any evidence of pericyte degenerative changes and suggest that pericyte degeneration analogous to that associated with human diabetic microangiopathy does not occur in this experimental animal model.     

Effectiveness of topical bevacizumab in bilateral primary lipid keratopathy

CASE REPORT: A 75-year-old man with bilateral idiopathic lipid keratopathy underwent a penetrating keratoplasty in the left eye. One month later, there was deep corneal neovascularisation extending across the bed and the graft-host interface, with a whitish opacity surrounding the vessels. Topical bevacizumab (25mg/mL) was administered 4 times daily for 2 months with partial regression of corneal neovascularization. DISCUSSION: Topical bevacizumab may be useful in preventing a recurrence of lipid deposition after penetrating keratoplasty in patients with bilateral primary lipid keratopathy, although its long-term efficacy needs to be assessed.     

角膜碱烧伤后大鼠角膜新生血管的细胞学研究

背景微血管壁由周细胞和血管内皮细胞组成，以往人们对血管系统的研究主要集中于血管内皮细胞，而对血管周细胞的研究较少。目的观察角膜碱烧伤后新生血管形成过程中的血管内皮细胞和周细胞的变化，探讨周细胞对新生血管生长的影响。方法36只健康成年SPF级sD大鼠，浸有1mol／LNaOH的4mm自制涂药器放置于右眼角膜中央3s，制作碱烧伤动物模型，3只正常sD大鼠作为正常对照。分别于角膜烧伤后1、2、3、5、7、10d各处死6只大鼠获取平行于角膜缘的环形切片，采用双重免疫荧光染色法检测CD31和α—SMA在角膜组织的表达，以分别评估血管内皮细胞和周细胞的分布，观察角膜碱烧伤后二者在角膜组织中的动态变化。光学显微镜下分别计数抗Ot—SMA阳性的周细胞数和抗CD31阳性的内皮细胞数，以二者的比值作为周细胞包裹指数（PCI），定量分析角膜碱烧伤后新生血管对周细胞的募集情况。结果角膜碱烧伤后1dCD31即可在角膜浅基质层表达，呈红色荧光，随着角膜碱烧伤时间的延长，CD31在角膜组织中的表达逐渐增加并进入深基质层，至角膜碱烧伤后7d表达的细胞数减少。呈绿色荧光的α一SMA在角膜碱烧伤2d于角膜浅基质层表达，在实验观察期内表达量均少于CD31，至碱烧伤后7d，残留的CD31阳性细胞被α—SMA阳性细胞包裹，未被包裹的CD31阳性细胞多数发生消退。碱烧伤后1、2、3、5、7、10d的PCI分别为0、16．07％、11．95％、43．84％、73．97％、86．21％，呈现递增的趋势。结论大鼠角膜碱烧伤后周细胞对血管内皮细胞的包绕可能对新生血管的成熟与稳定发挥重要作用。     

Immunohistochemical localization of vascular endothelial growth factor, transforming growth factor alpha, and transforming growth factor beta1 in human corneas with neovascularization

PURPOSE: To analyze presence and distribution of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)alpha, and TGFbeta1 in human corneas with neovascularization due to different corneal diseases. METHODS: Indirect immunohistochemistry for VEGF, TGFalpha, and TGFbeta1, was performed on paraffin-embedded corneas obtained by keratoplasty. Corneas from each of the four main groups of histopathologic diagnoses associated with corneal neovascularization were analyzed (scarring after keratitis, graft rejection/insufficiency, acute necrotizing keratitis, scarring after mechanical/chemical injury). Subclassification of inflammatory infiltrates was done using immunohistochemistry for CD3 (T-lymphocytes) and CD68 (macrophages). RESULTS: The analyzed angiogenic factors were detectable in corneas from all four histopathologic groups in a similar distribution; capillary endothelial cells, stromal and intravascular inflammatory cells (T-lymphocytes, macrophages), and basal corneal epithelial cells stained positive for the tested angiogenic factors. CONCLUSION: The angiogenic factors VEGF, TGFalpha, and TGFbeta1 are detectable in human corneas with neovascularization. Their distribution is quite uniform in different corneal diseases, resulting in corneal angiogenesis. An antiangiogenic therapy inhibiting corneal neovascularization by antagonizing angiogenic factors would have to counteract several angiogenic factors.     

Gene-based antiangiogenic applications for corneal neovascularization

Corneal avascularity is maintained by angiogenic privilege, an active process involving the production of higher level of angiostatic factors to offset the effect of angiogenic factors. A wide range of pathological insults to the cornea can disrupt this intricate equilibrium and promote angiogenesis and corneal neovascularization with resultant visual impairment. Corneal neovascularization is also a major risk factor for graft failure after keratoplasty. Current treatment options for corneal neovascularization are restricted by limited efficacy, adverse effects, and a short duration of action. The unique anatomical position and relative immune privilege of cornea make it an ideal tissue for gene-based therapies. Gene transfer vectors have been used to deliver or target genes involved in the pathogenesis of corneal neovascularization in animal models. Several proangiogenic and antiangiogenic factors have been targeted and assessed in experimentally induced corneal neovascularization. Antisense oligonucleotides targeting corneal neovascularization have entered human clinical trials and have not required vector delivery systems. The emergence of these RNA-based strategies heralds a new era in the management of corneal neovascularization and ocular therapeutics.     

Inhibition of experimental corneal neovascularisation by bevacizumab (Avastin)

AIM: To evaluate the effect of topically administered bevacizumab (Avastin) on experimental corneal neovascularisation in rats. METHODS: Silver nitrate sticks (75% silver nitrate, 25% potassium nitrate) were used to perform chemical cauterisation on the corneas of 16 eyes from 16 male Long Evans rats. For the following 7 days, the 10 eyes in the treatment group were instilled with bevacizumab 4 mg/ml drops twice daily, whereas the 6 eyes in the control group received placebo (normal saline drops twice daily). Digital photographs of the cornea were analysed to determine the area of cornea covered by neovascularisation as a percentage of the total corneal area. RESULTS: In the bevacizumab-treated eyes, neovascularisation covered, on average, 38.2% (15.5%) (mean (SD)) of the corneal surface compared with 63.5% (5.0%) in the control group (p<0.02, Mann-Whitney U test). CONCLUSION: Topically administered bevacizumab (Avastin) at a concentration of 4 mg/ml limits corneal neovascularisation following chemical injury in the male Long Evans rat model.     

Standardized semiquantitative analysis of corneal neovascularization using projected corneal photographs--pilot study after perforating corneal keratoplasty before immune reaction

BACKGROUND: A semiquantitative scheme for analysis of corneal neovascularization using projected corneal photographs is demonstrated and tested in a pilot study to analyze occurrence of corneal neovascularization in patients after perforating keratoplasty which subsequently developed transplant rejection. METHODS: Corneal photographs on the slit lamp with diffuse frontal illumination were obtained in a standardized technique. Slides were projected with 100 x magnification and analyzed twice with a 2 months interval. Corneal vessels were graded by two independent observers in each of 12 corneal sectors in a standardized fashion (grade 0: no vessels beyond limbus, 1: vessels between limbus and outer end of a double-running diagonal suture; 2: vessels between outer suture end and graft-host junction; 3: vessels reaching graft-host junction; 4: vessels within donor cornea). All patients with endothelial graft rejection of the prospective Erlangen non-high-risk keratoplasty study were included in a pilot study (1/1997-6/2000: 13 of 325; 4%). One patient without photographs available was excluded. Corneal photographs taken prior to surgery (n = 10), at the last 3 monthly-routine control before (10), at rejection episode (12) and one year later (10) were evaluated for corneal neovascularization. RESULTS: Interobserver correlation at the two assessments was 0.79 and 0.86 (Kendall's Tau B). Correlation between the assessments at the two analyses 2 months apart was 0.8. New vessels with diameter up to 6 microns can be detected. 8 of 12 analyzed patients (67%) with immune reaction after keratoplasty developed corneal neovascularization within 1 year after operation prior to transplant rejection in at least one corneal sector (2.1 +/- 1.9 sectors; 1-6). At time of rejection, new vessels reached the graft-host junction in 2 patients, in 1 patient vessels grew into the donor cornea, whereas in 8 the vessels were seen beyond the outer suture end without reaching host-graft junction (grade I: 1 patient). New vessels usually pointed to the outer suture ends of the double-running suture. CONCLUSIONS: Development of corneal neovascularization e.g. after keratoplasty can be assessed reliably using projected slides of corneal photographs at 100 x magnification. This method has the advantage of being more objective, precise and available compared to simple evaluation at the slit lamp. Postkeratoplasty corneal neovascularization seems to be common in non-high-risk eyes later developing transplant rejection. However, new vessels usually do not reach the host-graft junction. Whether neovascularization after keratoplasty demonstrates a risk factor for subsequent transplant rejection remains to be analyzed in a greater study.     

Degenerated intramural pericytes ('ghost cells') in the retinal capillaries of diabetic rats.

One of the earliest histopathological signs of diabetic retinopathy is a selective loss of intramural pericytes from retinal capillaries. In the present study, the retinal vessels of rats with streptozotocin-induced diabetes (STZ Wistar) and rats with genetically-induced insulin dependent diabetes mellitus (BB Wistar) and non-insulin dependent diabetes mellitus (SHR/N-corpulent) were examined after 6 to 8 months duration for diabetes-related retinal microangiopathies. The SHR/N-corpulent (cp) rats were fed a 54% sucrose diet, whereas the STZ Wistar and BB Wistar rats were fed laboratory chow for 32 to 36 weeks. In all the diabetic rats, the retinal capillaries in enzyme-digested flat mounts exhibited an increase in periodic-acid-Schiff (PAS) staining and loss of pericytes compared to their respective euglycemic controls. Pericyte "ghosts", like those defined in human diabetes as intramural pockets lacking normal cell contents, were documented by high resolution micrographs in all the diabetic rats. Endothelial cell proliferation, capillary dilation, and varicose loop formation were noted in some of the diabetic rats. Hence, similar capillary lesions were found in very different groups of diabetic rats. The findings suggest that a chronic high tissue concentration of glucose is the underlying factor which triggers pathogenesis in the pericyte. Hyperglycemia-induced activation of endogenous aldose reductase of the polyol pathway is probably the initial insult, but other factors such as advanced glycosylation products may affect the final outcome.     

角膜移植后角膜新生淋巴管与新生血管间的关联

目的:研究角膜移植后角膜新生淋巴管与新生血管间关联。方法:人角膜取自行二次角膜移植的19名患者。5核苷酸酶-碱性磷酸酶(5-nase-Alkaline phosphatase,5-NA-ALP)双重酶组织化学染色及淋巴内皮细胞受体(lymphatic vessel endothelial receptor,LYVE-1)、内皮细胞黏附因子-1(platelet endothelial cell adhesion modecule-1,PECAM-1)双重免疫组化法标记角膜中的新生血管和淋巴管,并进行淋巴管计数(lymphatic vessels counting,LVC)和血管计数(blood vessels counting,BVC),比较BVC与LVC之间的关联。结果:角膜中存在角膜新生血管12例(63%),存在角膜新生淋巴管5例(26%)。角膜新生淋巴管仅出现在血管化角膜中。角膜移植后BVC与LVC间呈显著性正相关(r=0.725;P<0.01)。结论:人角膜移植后角膜新生淋巴管与新生血管之间存在密切关联。     

Endothelin-1 and ETA/ETB receptor protein and mRNA: expression in normal and vascularized human corneas

PURPOSE: Neovascularization of the cornea causes blindness and increases the risk of immune rejections after keratoplasty. The purpose of this study was to investigate involvement of the potent angiogenic growth factor endothelin (ET)-1 and its receptors, ETA and ETB, in corneal neovascularization. METHODS: ET-1, ETA, and ETB receptor protein expression was evaluated in nonvascularized and vascularized human corneas by immunohistochemistry. Epithelial ET-1 protein expression of both groups was compared using a semiquantitative scoring system. Double immunofluorescence was used to colocalize ETA and ETB receptor with CD31. In situ hybridization and immunoelectron microscopy analyzed ET-1 and its receptors in normal and vascularized corneas. RESULTS: Nonvascularized corneas displayed ET-1 and ETA/ETB receptor protein and mRNA in epithelial and some corneal endothelial cells. ETA more than ETB receptors were expressed on some keratocytes. In vascularized corneas, ET-1 and ETA/ETB receptor expression was found in the endothelial lining of new blood vessels (as shown by CD31-colocalization). ET-1 protein expression was significantly increased in the epithelium of vascularized corneas (P < 0.001). Immunogold localized ET-1 and its receptors to the nuclear/perinuclear space and to the luminal side of endothelial cells of new blood vessels. CONCLUSIONS: In corneal neovascularization, ET-1 protein and mRNA expression is upregulated in epithelial cells. Together with ET-1, ETA, and ETB receptor expression on endothelial cells of ingrown new blood vessels, this points to an involvement of ET-1 and its receptors in corneal angiogenesis. As potent ETA and ETB receptors are available, the endothelin system may represent an additional target for corneal antiangiogenic therapy.     

半胱氨酸天冬氨酸蛋白酶在视网膜毛细血管周细胞凋亡中的活性及意义

目的探讨糖基化终产物（AGE）在糖尿病视网膜病变（DR）发生中的作用。方法培养的牛视网膜毛细血管周细胞分别与不同浓度（0．47、1．88、7．50μmol／L）的AGE共同培养4d后,分别检测细胞凋亡、半胱氨酸天冬氨酸蛋白酶（caspase-3）活性及caspase-3抑制剂Z—DEVD-fmk对周细胞凋亡及凋亡调节基因Bcl-2／Bax比率的影响。结果AGE能以剂量依赖的方式诱导培养的牛视网膜毛细血管周细胞凋亡（r=0．867,P〈0．01）、增加细胞内caspase-3的活性,而选择性caspase-3抑制剂Z—DEVD—fmk能明显抑制AGE作用下的周细胞凋亡及提高凋亡调节基因Bcl-2／Bax的比率。结论凋亡是DR中视网膜毛细血管周细胞选择性丧失的机制之一,caspase-3活性的增加是AGE诱导周细胞发生凋亡的关键因素。     

Corneal lymphangiogenesis correlates closely with hemangiogenesis after keratoplasty

AIM: To examine the relationship between corneal lymphangiogenesis and hemangiogenesis after keratoplasty. · METHODS: Nineteen human corneas were obtained from 19 patients undergoing a second corneal transplantation in Zhongshan Ophthalmic Center in 2005. Blood and lymphatic vessels in human transplanted corneas were identified by lymphatic vessel endothelial receptor (LYVE-1) and platelet endothelial cell adhesion modecule-1 (PECAM-1) immunohi- stochemistry, and double enzyme-histochemistry; then the association of corneal blood vessel counting (BVC) with lymphatic vessel counting (LVC) was examined. · RESULTS: Corneal hemangiogenesis was present in 12 cases (63%), and lymphangiogenesis occurred in 5 cases (26%) human transplanted corneas. In addition, corneal lymphangiogenesis was only present in vascularized corneas. LVC was strongly and positively correlated with BVC(r=0.725, P <0.01). · CONCLUSION: Corneal lymphangiogenesis develops after keratoplasty and strongly associates with hemangiogenesis.     

Inhibition of rat corneal angiogenesis by 16-kDa prolactin and by endogenous prolactin-like molecules.

PURPOSE: The cornea is an avascular organ, where induction of new blood vessels involves the turn-on of proangiogenic factors and/or the turn-off of antiangiogenic regulators. Prolactin (PRL) fragments of 14 kDa and 16 kDa bind to endothelial cell receptors and inhibit angiogenesis. This study was designed to determine whether antiangiogenic PRL-like molecules are involved in cornea avascularity. METHODS: Sixteen-kDa PRL and basic fibroblast growth factor (bFGF) or anti-PRL antibodies were placed into rat cornea micropockets and neovascularization evaluated by the optical density associated with capillaries stained by the peroxidase reaction and by the number of vessels growing into the implants. Prolactin receptors in corneal epithelium were investigated by immunocytochemistry. RESULTS: bFGF induced a dose-dependent stimulation of corneal neovascularization. This effect was inhibited by coadministration of 16-kDa PRL, as indicated by a 65% reduction in vessel density and a 50% decrement in the incidence of angiogenic responses. Corneal angiogenic reactions of different intensities were induced by implantation of polyclonal and monoclonal anti-PRL antibodies. Corneal epithelial cells were labeled by several anti-PRL receptor monoclonal antibodies. CONCLUSIONS: These findings show that exogenous 16-kDa PRL inhibits bFGF-induced corneal neovascularization and suggest that PRL-like molecules with antiangiogenic actions function in the cornea. PRL receptors in the corneal epithelium may imply that PRL in the cornea derives from lacrimal PRL internalized through an intracellular pathway. These observations are consistent with the notion that members of the PRL family are potential regulators of corneal angiogenesis.     

Absence of blood and lymphatic vessels in the developing human cornea

PURPOSE: The normal human cornea is devoid of both blood and lymphatic vessels and actively maintains this avascularity (corneal angiogenic privilege). Whether and when corneal angiogenic privilege is achieved during development is unknown. METHODS: This study analyzed whether the cornea is primarily devoid of both blood and lymphatic vessels during intrauterine development or whether secondary regression of pre-existing vessels occurs before delivery. Indirect double immunohistochemistry was performed on 4-microm serial pupil-optic disc sections of paraffin-embedded human eyes stillborn at gestational ages of 17 to 41 weeks with antibodies against von Willebrand factor (vWF; factor VIII-associated antigen) as a panendothelial marker and with antibodies against lymphatic vessel endothelial hyaluronate receptor 1 (LYVE1) as a marker specific for lymphatic vascular endothelium. RESULTS: Human corneas were devoid of both vWF+++/LYVE-1(-) blood vessels and vWF+/LYVE-1+++ lymphatic vessels at all time-points analyzed. In contrast, there were numerous blood and lymphatic vessels detectable in the adjacent conjunctiva. CONCLUSION: The normal human cornea is primarily avascular and devoid of both blood and lymphatic vessels. Corneal angiogenic privilege is already achieved very early during fetal intrauterine development. This suggests early and strong expression of both antiangiogenic and antilymphangiogenic factors in the human cornea during development.