Anders Kvanta

Biomechanical regulation of blood vessel growth during tissue vascularization

Formation of new vessels in granulation tissue during wound healing has been assumed to occur solely through sprouting angiogenesis. In contrast, we show here that neovascularization can be accomplished by nonangiogenic expansion of preexisting vessels. Using neovascularization models based on the chick chorioallantoic membrane and the healing mouse cornea, we found that tissue tension generated by activated fibroblasts or myofibroblasts during wound contraction mediated and directed translocation of the vasculature. These mechanical forces pulled vessels from the preexisting vascular bed as vascular loops with functional circulation that expanded as an integral part of the growing granulation tissue through vessel enlargement and elongation. Blockade of vascular endothelial growth factor receptor-2 confirmed that biomechanical forces were sufficient to mediate the initial vascular growth independently of endothelial sprouting or proliferation. The neovascular network was further remodeled by splitting, sprouting and regression of individual vessels. This model explains the rapid appearance of large functional vessels in granulation tissue during wound healing.

A prospective study on intravitreal bevacizumab (Avastin®) for neovascular age‐related macular degeneration of different durations

Purpose:  Choroidal neovascularization (CNV) accounts for 85–90% of severe visual impairment in age‐related macular degeneration (AMD). Vascular endothelial growth factor (VEGF) is a major factor mediating angiogenesis, and VEGF inhibitors have become a new treatment modality. In this prospective study, we used bevacizumab (Avastin®), a recombinant monoclonal antibody to VEGF, to treat neovascular AMD.

Benefit from Bevacizumab for Macular Edema in Central Retinal Vein Occlusion: Twelve-Month Results of a Prospective, Randomized Study

PURPOSE To evaluate the efficacy of intraocular injections with bevacizumab over 12 months in patients with macular edema (ME) secondary to central retinal vein occlusion (CRVO). DESIGN A prospective study including a randomized 6-month, sham injection-controlled, double-masked clinical trial followed by a 6-month open-label extension. PARTICIPANTS Sixty patients with ME secondary to CRVO. METHODS At baseline, patients were randomized 1:1 to receive intraocular injections of bevacizumab or sham injections every 6 weeks for 6 months. From month 6, all patients received intraocular injections of bevacizumab every 6 weeks for 6 months. MAIN OUTCOME MEASURES The primary outcome measure was the proportion of patients gaining at least 15 letters at 12 months. Secondary outcome measures included mean change from baseline best-corrected visual acuity (BCVA), change in foveal thickness, and development of neovascular glaucoma. RESULTS At the end of follow-up, 18 of 30 patients (60.0%) in the bevacizumab/bevacizumab (bz/bz) group had gained ≥ 15 letters compared with 10 of 30 patients (33.3%) in the sham/bevacizumab (sh/bz) group (P < 0.05). The BCVA improved by 16.0 letters at 12 months in the bz/bz group compared with 4.6 letters in the sh/bz group (P < 0.05). In an unplanned retrospective analysis, patients aged >70 years had a significantly worse outcome when receiving delayed treatment, losing 1.4 letters (95% confidence interval [CI], -9.7 to 8.4) in the sh/bz group compared with a gain of 20.1 letters (95% CI, 13.9-26.3) in the bz/bz group in patients aged <70 years (P < 0.003). The mean decrease in central retinal thickness (CRT) was 435 μm in the bz/bz group compared with 404 μm in the sh/bz group (P = not significant). No patients developed iris rubeosis during the 6-month open-label extension period. There were no events of endophthalmitis, retinal tear, or retinal detachment during the 12-month treatment period. No serious nonocular adverse events were reported. CONCLUSIONS Intraocular injections of bevacizumab given every 6 weeks for 12 months improve visual acuity (VA) and reduce ME significantly. Patients receiving delayed treatment have a limited visual improvement. FINANCIAL DISCLOSURE(S) Proprietary or commercial disclosure may be found after the references.

Expression and regulation of vascular endothelial growth factor in choroidal fibroblasts

Subretinal neovascularization is a severe sight-threatening complication into age-related macular degeneration. Previous immunohistochemical studies on surgically removed neovascular membranes have revealed that these membranes, in addition to the neovascular stroma, are comprised of several different cell types such as retinal pigment epithelial (RPE) cells, choroidal fibroblasts and vascular endothelial cells. Since vascular endothelial growth factor (VEGF) potently and specifically induces angiogenesis it was investigated whether VEGF is expressed and/or inducible in choroidal fibroblasts and RPE cells. Choroidal fibroblasts and RPE cells were isolated from human adult post-mortem eyes and expression of VEGF mRNA and protein was measured. By using Northern blotting, both choroidal fibroblasts and RPE cells were found to express VEGF mRNA at low levels. In order to examine whether this VEGF expression was further inducible, the intracellular effector enzyme protein kinase C was activated by phorbol esters. This activation resulted in a prominent increase in VEGF mRNA in choroidal fibroblasts, but not in RPE cells, with a maximal increase detected after 6 h. Elevation of intracellular cyclic AMP levels by forskolin had no clear effect on VEGF mRNA in either cell type. Stimulation with interleukin-1, transforming growth factor beta, tumour necrosis factor alpha and platelet derived growth factor was tested to see if VEGF expression is cytokine inducible. Both interleukin-1 and transforming growth factor beta induced VEGF expression in choroidal fibroblasts although with different time courses. Whereas the transforming growth factor beta effect was transient the interleukin-1 effect was sustained for at least 48 h. None of the cytokines tested affected VEGF expression in RPE cells. By using Western blotting, it was further found that stimulation with interleukin-1 induced VEGF protein expression in choroidal fibroblasts but not in RPE cells. In conclusion, choroidal fibroblasts respond by elevated VEGF mRNA levels after phorbol ester, interleukin-1 and transforming growth factor beta stimulation and elevated VEGF protein levels after phorbol ester and interleukin-1 stimulation suggesting that choroidal fibroblasts may be target cells for increased VEGF synthesis secondary to paracrine cytokine production.

Bevacizumab for Macular Edema in Central Retinal Vein Occlusion: A Prospective, Randomized, Double-Masked Clinical Study

PURPOSE To evaluate the efficacy of intraocular injections with bevacizumab in patients with macular edema (ME) secondary to central retinal vein occlusion (CRVO). DESIGN Prospective, randomized, sham injection-controlled, double-masked clinical trial. PARTICIPANTS Sixty patients with ME secondary to CRVO. METHODS At baseline, patients were randomized 1:1 to receive intraocular injections of bevacizumab or sham injections every 6 weeks for 6 months. MAIN OUTCOME MEASURES The primary outcome measure was the proportion of patients gaining at least 15 letters at 6 months. Secondary outcome measures included mean change from baseline best-corrected visual acuity (BCVA), foveal thickness, and neovascular glaucoma. RESULTS At the end of follow-up, 18 of 30 patients (60.0%) in the study group had gained ≥15 letters compared with 6 of 30 patients (20.0%) in the control group (P=0.003). The BCVA improved by 14.1 letters at 24 weeks compared with a decrease of 2.0 letters in the control group (P < 0.003). The mean decrease in central retinal thickness (CRT) was significantly greater in the study group (426 μm) than in the control group (102 μm) at all time points up to week 24 (P < 0.001). No residual edema, defined as CRT <300 μm at 24 weeks, was found in 26 of 30 patients (86.7%) in the treatment group compared with 6 of 30 patients (20%) in the control group (P < 0.001). In the sham group, 5 of 30 patients (16.7%) had developed iris rubeosis at week 24. No patients in the study group had rubeosis at week 24 (P=0.052). There were no events of endophthalmitis, retinal tear, or retinal detachment during the 24-week treatment period. No serious non-ocular adverse events were reported. CONCLUSIONS Intraocular injections of bevacizumab given every 6 weeks for 6 months improve visual acuity (VA) and reduce ME significantly compared with sham. FINANCIAL DISCLOSURE(S) Proprietary or commercial disclosure may be found after the references.

Matrix metalloproteinase (MMP) expression in experimental choroidal neovascularization

PURPOSE Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that degrade basement membrane and extracellular matrix proteins. To gain information on the possible role of MMPs in choroidal neovascularization (CNV), we have analyzed the mRNA expression of MMP-2 and MMP-9, two forms of MMPs implicated in ocular neovascularization, in a rat model. METHODS Choroidal neovascularization was induced in pigmented rats by krypton laser photocoagulation of the fundus whereafter eyes were enucleated at 1, 3, 5, 7, 10 and 60 days. Antisense and sense riboprobes were generated using DNA complementary to MMP-2 and MMP-9, and mRNA expression was analyzed using in situ hybridization. RESULTS In the untreated eyes MMP-2 mRNA expression was weakly detected in cells within the choroid. In laser-treated eyes MMP-2 mRNA expression was markedly increased and mainly localized to macrophage-like and retinal pigment epithelial (RPE)-like cells invading the choroid, subretinal space and inner retina. This increase in MMP-2 mRNA expression peaked at day 10 whereafter a decline was detected. MMP-9 mRNA expression was low in untreated eyes and did not increase following laser treatment. CONCLUSION The results show that MMP-2 mRNA expression is increased in experimental CNV, and support of a role for MMP-2 in the development of CNV in age-related macular degeneration.

Further evidence that propentofylline (HWA 285) influences both adenosine receptors and adenosine transport

Summary— We have examined the actions of a novel xanthine derivative, propentofylline (HWA 285), that has been shown to protect against ischemic brain damage in rats and gerbils, on adenosine receptors (A1 and A2), and on adenosine transporters using several techniques, cells and tissues. Propentofylline and its hydroxylated metabolite A 72 0287 were about 20 times less potent than theophylline in displacing A1‐agonist binding to membranes from rat cortex, and A1‐antagonist binding to whole DDT, MF‐2 smooth muscle cells. A1‐agonist binding to adenosine A1‐receptors in several brain structures was inhibited in a concentration‐dependent manner by A 72 0287 and propentofylline as judged by quantitative autoradiography (IC50‐values 300–600 μM in eg striatum and in cortex layer IV). In two functional assays. A1‐receptor mediated effects were blocked by propentofylline. A1‐receptor‐mediated inhibition of cyclic AMP accumulation was virtually abolished by 100 μM propentofylline. The A1‐receptor‐mediated inhibition of evoked acctylcholine release was also reduced by propentofylline, but in this case the effect is not due exclusively to adenosine receptor antagonism but also to another action since the presynaptic inhibitory effect of carbachol was also inhibited. Adenosine A2‐receptors were also antagonized by propentofylline as judged by a concentration‐dependent antagonism of A2‐agonist‐induced cAMP accumulation in human T‐leukemia cells (possessing putative A2b‐receptors; pA2‐value 180 μM compared to 0.26 μM for 8‐cpt), and in PC‐12 cells (possessing putative A2a‐receptors, K1‐value 365 μM). Finally, adenosine transporters were affected by propentofylline and A 72 0287. Thus, [3H]‐nitrobenzylthioinosine‐binding to guinea‐pig cardiac membranes was blocked by propentofylline or A 72 0287 (Ki 270 μM). The present results show that propentofylline and its hydroxylated metabolite can influence adenosine mechanisms in a multitude of ways. How these different actions may contribute to the ability of propentofylline to reduce the magnitude of ischemic damage is discussed.

Inhibition of VEGF secretion and experimental choroidal neovascularization by picropodophyllin (PPP), an inhibitor of the insulin-like growth factor-1 receptor.

INTRODUCTION Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration (AMD) and a leading cause of vision loss. Along with other angiogenic factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and its receptor, IGF-1R, have been implicated in CNV. PURPOSE A prior study has shown that the cyclolignan picropodophyllin (PPP) efficiently blocks the insulin-like growth factor-1 receptor (IGF-1R) activity and causes cell death in uveal melanoma cell lines and in an in vivo model. In this study we investigated the effect of PPP on VEGF expression, both in vitro and in vivo, and whether this effect has antiangiogenic consequences in a murine CNV model. METHODS C57BL/6J mice with laser-induced CNVs were treated with PPP. Effects on CNV area were assayed by image analysis. VEGF levels in the choroid and retinal pigment epithelial cells (ARPE-19) were measured by Western blot or ELISA. Transcriptional activation of the VEGF promoter was determined by luciferase reporter gene assay. RESULTS Mice treated with PPP, administered intraperitoneally or orally, showed a 22% to 32% (P = 0.002) decrease in CNV area. Furthermore, VEGF levels in the choroid were significantly reduced. In cultured ARPE-19 cells, IGF-1 was shown to increase VEGF secretion. This increase was completely blocked by PPP. PPP reduced the level of transcriptional activity of the VEGF promoter. CONCLUSIONS PPP reduces IGF-1-dependent VEGF expression and CNV in vivo. Accordingly, IGF-1R inhibitors may be useful tools in the treatment of conditions associated with CNV, including neovascular AMD.

Expression and Secretion of Transforming Growth Factor-β in Transformed and Nontransformed Retinal Pigment Epithelial Cells

The expression and secretion of different isoforms of transforming growth factor-beta (TGF beta) were examined in cultured transformed and nontransformed human retinal pigment epithelial (RPE) cells. Transformed RPE cells were found to express high levels of TGF beta 1 mRNA, low levels of TGF beta 3 mRNA but no detectable TGF beta 2 mRNA. If the cells were grown under serum-free conditions the expression of TGF beta increased. The mRNA expression was accompanied by secretion of TGF beta 1 (but not TGF beta 2) protein into the culture media. By comparison, nontransformed RPE cells were found to secrete similar amounts of TGF beta as transformed cells but predominantly secreted TGF beta 2. The secretion of TGF beta from both transformed and nontransformed RPE cells increased if the cells were grown without serum. In conclusion, the results show that TGF beta is expressed and secreted by transformed and nontransformed human RPE cells and that this expression and secretion are regulated by the presence or absence of exogenous factors.

Stimulation of T-cells with OKT3 antibodies increases forskolin binding and cyclic AMP accumulation

It has recently been shown that elevation of cAMP by adenosine receptor stimulation may be potentiated by stimulation of the T-cell receptor/CD3 complex on human T-cells with the monoclonal antibody OKT3, and that this is mimicked by activation of protein kinase C [Kvanta, A. et al. (1989) Naunyn-Schmeidebergs Arch. Pharmac. 340, 715-717]. In this study the diterpene forskolin, which binds to and activates the adenylate cyclase, has been used to examine further how the CD3 complex may influence the adenylate cyclase pathway. Stimulation with OKT3 alone was found to cause a small dose-dependent increase in basal cAMP accumulation. When combining OKT3 with a concentration of forskolin (10 microM), which by itself had little effect on the cyclase activity, the cAMP accumulation was markedly potentiated. This potentiation was paralleled by an increase in [3H]forskolin binding to saponine permeabilized Jurkat cells from 24 to 41 fmol/10(6) cells. The OKT3 effect on cAMP was blocked by chelating extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA and also by W-7, an inhibitor of calmodulin, but was unaffected by H-7, an inhibitor of protein kinase C. Even though OKT3 caused an increase in inositolphosphate turnover, and activated protein kinase C, neither phorbol 12,13 dibutyrate (PDBu) nor the Ca2(+)-ionophore A23187 could mimic the OKT3 effect, whereas a combination of PDBu and A23187 at high concentrations could potentiate forskolin stimulated cyclase activity. Together, these results indicated that stimulation of the CD3 complex could influence the adenylate cyclase by two different mechanisms, one involving activation of protein kinase C and another which does not.