Regulation of Angiogenesis : Role of R-Ras, furin and syndecan 4 in retinal angiogenesis

Angiogeneesi, eli verisuonten uudismuodostus on tärkeää normaalin kehityksen ja kudoksen tasapainon kannalta. Useissa sairauksissa muutoin tarkkaan säädellyt mekanismit järkkyvät, mikä voi johtaa verisuonten hallitsemattomaan kasvuun. Patologista uudisverisuonitusta esiintyy useissa näköa heikentävissä ja sokeuttavissa silmänpohjan sairauksissa sekä useissa syövissä. Tiedetään, että verisuonten endoteelikasvutekijä (VEGF) on merkittävassä roolissa edistämässä haitallista verisuonten kasvua, ja sen estäjiä käytetäänkin hoitona useissa neovaskulaarisissa sairauksissa. Nämä hoidot eivät aina kuitenkaan ole tarpeeksi tehokkaita, ja uusia tehokkaampia ja spesifisempia hoitomuotoja tarvitaan. Tämän takia angiogeneesin liittyvien tapahtumien parempi molekulaarinen tietämys on tarpeen. Tämän väitöskirjan tavoitteena oli tutkia eri geenien vaikutusta angiogeneesiin ja läytää uusia angiogeneesin säätelyyn osallistuvia tekijöitä. Väitöskirja koostuu neljästä osatyöstä, jotka käsittelevät angiogeneesia retinan kehityksen aikana sekä osana silmäsairauksia. Osatöissä on suurelta osin käytetty kokeellisia hiirimalleja, jotka mallintavat iskeemisia retinopatioita. Lisäksi käytössä on ollut lasiaisenpoistoleikkauksen yhteydessä saatuja potilasnäytteitä proliferatiivista diabeettista retinopatiaa ja verkkokalvon verisuonitukosta sairastavailta potilailta. Ensimmäisessä osatyössä Rras geenin roolia tutkittiin iskeemisen retinopatian angiogeneesissa, ja todettiin geenin merkittavä osallisuus verkkokalvon verisuonten läpäisevyyden säätelyssä. Meyloidisolujen furin geenin merkitystä verkkokalvon verisuonten kasvuun tutkittiin toisessa osatyössä ja havaittiin sen edistävän verisuonten kasvua. Kolmannessa osatyössä kokeellisesta iskeemisestä verkkokalvon angiogeneesimallista tehtiin tähän mennessä laajin hiiren verkkokalvon proteomin kartoittava seulontatutkimus. Tutkimuksessa löydettiin proteiineja, jotka mahdollisesti osallistuvat verkkokalvon haitallisen uudisverisuonitukseen muodostumiseen hiirimallissa sekä ihmisen iskeemisissä retinopatioissa. Viimeisessä osatyössä tutkittiin syndekaani-4 -geenin (Sdc4) merkitystä angiogeneesille useissa eri malleissa ja havaittiin SDC4:n merkittävä rooli VEGFA-välitteisen patologisen verisuonituksen muodostumisessa ja VE-kadheriinin toiminnassa. SDC4 terapeuttista potentiaalia testattiin kokeellisessa silmänpohjan ikärappeumamallissa, ja todettiin SDC4 toiminnan eston olevan yhtä tehokas kuin ikärappeuman hoidossa kliinisessä käytössä oleva lääke. Tutkimuksessa saadut tulokset lisäävät tietämystä angiogeneesiin vaikuttavista tekijöistä. Ne voivat olla hyödyllisiä kehitettäessä uusia potentiaalia lääkkeitä sairauksiin, joihin liittyy haitallinen verisuonten uudismuodostus.Angiogenesis, the formation of new blood vessels, is important for normal development and the maintenance of tissue homeostasis. However, in many diseases, the events that are normally tightly controlled become disrupted, which can lead to aberrant and extensive growth of the blood vessels. Pathological angiogenesis is a key feature in many vision-threatening eye diseases and in cancers. Vascular endothelial growth factor (VEGF) is the main driver of pathological angiogenesis and VEGF- inhibitors are commonly used as a treatment for neovascular diseases. However, the therapies are not always sufficiently effective, and resistance to them can develop. Thus, more effective and specific therapeutics are needed. For this reason, the better understanding of the molecular mechanisms behind the pathologies is important. The main aim of this dissertation was to study the effect of different genes on the formation of new blood vessels and to identify new factors that contribute to the regulation of angiogenesis. The dissertation consists of four studies that addressed the role of angiogenesis mainly in eye diseases. These studies made use of experimental models of retinopathies as well as samples from human patients with proliferative diabetic retinopathy and retinal vein occlusion. The first study investigated the role of R-Ras in the regulation of angiogenesis in ischemic retinopathy. We found that R-Ras was important for the maintenance of vascular stability. The same ischemic retinopathy model was used in the second study, in which we showed that the function of myeloid-specific furin for retinal angiogenesis plays a role in retinal revascularization. In the third study we performed the most comprehensive proteomic profiling of the oxygen-induced retinopathy model to date. We identified novel proteins that may drive the pathogenesis and neovascularization in the mouse retinopathy model. These proteins may have relevance to human retinopathy as well as new potential drug targets. The fourth study explored the role of syndecan 4 (SDC4) in angiogenesis using several angiogenesis models. We demonstrated that SDC4 is needed for VEGF-\- driven pathological angiogenesis and VE-cadherin trafficking. The therapeutic potential of SDC4 inhibition was tested in an experimental model of wet age-related macular degeneration (AMD), and we found that it was as effective as a drug commonly used in the clinic for the treatment of wet AMD. The results of these studies improve the knowledge of the factors that affect angiogenesis. These results could be useful in the future development of new drugs for diseases that involve pathological angiogenesis

Selective targeting and tissue penetration to the retina by a systemically administered vascular homing peptide in oxygen induced retinopathy (OIR)

Pathological angiogenesis is the hallmark of ischemic retinal diseases among them retinopa-thy of prematurity (ROP) and proliferative diabetic retinopathy (PDR). Oxygen-induced retinopathy (OIR) is a pure hypoxia-driven angiogenesis model and a widely used model for ischemic retinopathies. We explored whether the vascular homing peptide CAR (CARSKNKDC) which recognizes angiogenic blood vessels can be used to target the retina in OIR. We were able to demonstrate that the systemically administered CAR vascular homing peptide homed selectively to the preretinal neovessels in OIR. As a cell and tissue-penetrating peptide, CAR also penetrated into the retina. Hyperoxia used to induce OIR in the retina also causes bronchopulmonary dysplasia in the lungs. We showed that the CAR peptide is not targeted to the lungs in normal mice but is targeted to the lungs after hyperoxia-/hypoxia-treatment of the animals. The site-specific delivery of the CAR peptide to the pathologic retinal vasculature and the penetration of the retinal tissue may offer new opportunities for treating retinopathies more selectively and with less side effects.publishedVersionPeer reviewe

Resistance of R-Ras knockout mice to skin tumour induction

The R-ras gene encodes a small GTPase that is a member of the Ras family. Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it. R-Ras activity appears inhibitory towards tumour proliferation and invasion, and to promote cellular quiescence. Contrary to this, using mice with a deletion of the R-ras gene, we found that R-Ras facilitates DMBA/TPA-induced skin tumour induction. The tumours appeared in wild-type (WT) mice on average 6 weeks earlier than in R-Ras knockout (R-Ras KO) mice. WT mice developed almost 6 times more tumours than R-Ras KO mice. Despite strong R-Ras protein expression in the dermal blood vessels, no R-Ras could be detected in the epidermis from where the tumours arose. The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression. Thus, these data suggest that despite its characterised role in promoting cellular quiescence, R-Ras is pro-tumourigenic in the DMBA/TPA tumour model and important for the inflammatory response to DMBA/TPA treatment

Pathological Angiogenesis Requires Syndecan-4 for Efficient VEGFA-Induced VE-Cadherin Internalization

Objective: VEGFA (Vascular endothelial growth factor A) and its receptor VEGFR2 (vascular endothelial growth factor receptor 2) drive angiogenesis in several pathologies, including diabetic retinopathy, wet age-related macular degeneration, and cancer. Studies suggest roles for HSPGs (heparan sulfate proteoglycans) in this process, although the nature of this involvement remains elusive. Here, we set to establish the role of the HSPG SDC4 (syndecan-4) in pathological angiogenesis. Approach and Results: We report that angiogenesis is impaired in mice null for SDC4 in models of neovascular eye disease and tumor development. Our work demonstrates that SDC4 is the only SDC whose gene expression is upregulated during pathological angiogenesis and is selectively enriched on immature vessels in retinas from diabetic retinopathy patients. Combining in vivo and tissue culture models, we identified SDC4 as a downstream mediator of functional angiogenic responses to VEGFA. We found that SDC4 resides at endothelial cell junctions, interacts with vascular endothelial cadherin, and is required for its internalization in response to VEGFA. Finally, we show that pathological angiogenic responses are inhibited in a model of wet age-related macular degeneration by targeting SDC4. Conclusions: We show that SDC4 is a downstream mediator of VEGFA-induced vascular endothelial cadherin internalization during pathological angiogenesis and a potential target for antiangiogenic therapies.acceptedVersionPeer reviewe

CD73 controls ocular adenosine levels and protects retina from light-induced phototoxicity

ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5'-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39(high)/CD73(low) processes and forming local "purinergic junctions" with CD39(low)/CD73(-) neuronal cell bodies and CD39(high)/CD73(-) retinal blood vessels. The relevance of the CD73-adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.</p

Selective Targeting and Tissue Penetration to the Retina by a Systemically Administered Vascular Homing Peptide in Oxygen Induced Retinopathy (OIR)

Pathological angiogenesis is the hallmark of ischemic retinal diseases among them retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR). Oxygen-induced retinopathy (OIR) is a pure hypoxia-driven angiogenesis model and a widely used model for ischemic retinopathies. We explored whether the vascular homing peptide CAR (CARSKNKDC) which recognizes angiogenic blood vessels can be used to target the retina in OIR. We were able to demonstrate that the systemically administered CAR vascular homing peptide homed selectively to the preretinal neovessels in OIR. As a cell and tissue-penetrating peptide, CAR also penetrated into the retina. Hyperoxia used to induce OIR in the retina also causes bronchopulmonary dysplasia in the lungs. We showed that the CAR peptide is not targeted to the lungs in normal mice but is targeted to the lungs after hyperoxia-/hypoxia-treatment of the animals. The site-specific delivery of the CAR peptide to the pathologic retinal vasculature and the penetration of the retinal tissue may offer new opportunities for treating retinopathies more selectively and with less side effects

Regulation of Angiogenesis : Role of R-Ras, furin and syndecan 4 in retinal angiogenesis

Angiogeneesi, eli verisuonten uudismuodostus on tärkeää normaalin kehityksen ja kudoksen tasapainon kannalta. Useissa sairauksissa muutoin tarkkaan säädellyt mekanismit järkkyvät, mikä voi johtaa verisuonten hallitsemattomaan kasvuun. Patologista uudisverisuonitusta esiintyy useissa näköa heikentävissä ja sokeuttavissa silmänpohjan sairauksissa sekä useissa syövissä. Tiedetään, että verisuonten endoteelikasvutekijä (VEGF) on merkittävassä roolissa edistämässä haitallista verisuonten kasvua, ja sen estäjiä käytetäänkin hoitona useissa neovaskulaarisissa sairauksissa. Nämä hoidot eivät aina kuitenkaan ole tarpeeksi tehokkaita, ja uusia tehokkaampia ja spesifisempia hoitomuotoja tarvitaan. Tämän takia angiogeneesin liittyvien tapahtumien parempi molekulaarinen tietämys on tarpeen. Tämän väitöskirjan tavoitteena oli tutkia eri geenien vaikutusta angiogeneesiin ja läytää uusia angiogeneesin säätelyyn osallistuvia tekijöitä. Väitöskirja koostuu neljästä osatyöstä, jotka käsittelevät angiogeneesia retinan kehityksen aikana sekä osana silmäsairauksia. Osatöissä on suurelta osin käytetty kokeellisia hiirimalleja, jotka mallintavat iskeemisia retinopatioita. Lisäksi käytössä on ollut lasiaisenpoistoleikkauksen yhteydessä saatuja potilasnäytteitä proliferatiivista diabeettista retinopatiaa ja verkkokalvon verisuonitukosta sairastavailta potilailta. Ensimmäisessä osatyössä Rras geenin roolia tutkittiin iskeemisen retinopatian angiogeneesissa, ja todettiin geenin merkittavä osallisuus verkkokalvon verisuonten läpäisevyyden säätelyssä. Meyloidisolujen furin geenin merkitystä verkkokalvon verisuonten kasvuun tutkittiin toisessa osatyössä ja havaittiin sen edistävän verisuonten kasvua. Kolmannessa osatyössä kokeellisesta iskeemisestä verkkokalvon angiogeneesimallista tehtiin tähän mennessä laajin hiiren verkkokalvon proteomin kartoittava seulontatutkimus. Tutkimuksessa löydettiin proteiineja, jotka mahdollisesti osallistuvat verkkokalvon haitallisen uudisverisuonitukseen muodostumiseen hiirimallissa sekä ihmisen iskeemisissä retinopatioissa. Viimeisessä osatyössä tutkittiin syndekaani-4 -geenin (Sdc4) merkitystä angiogeneesille useissa eri malleissa ja havaittiin SDC4:n merkittävä rooli VEGFA-välitteisen patologisen verisuonituksen muodostumisessa ja VE-kadheriinin toiminnassa. SDC4 terapeuttista potentiaalia testattiin kokeellisessa silmänpohjan ikärappeumamallissa, ja todettiin SDC4 toiminnan eston olevan yhtä tehokas kuin ikärappeuman hoidossa kliinisessä käytössä oleva lääke. Tutkimuksessa saadut tulokset lisäävät tietämystä angiogeneesiin vaikuttavista tekijöistä. Ne voivat olla hyödyllisiä kehitettäessä uusia potentiaalia lääkkeitä sairauksiin, joihin liittyy haitallinen verisuonten uudismuodostus.Angiogenesis, the formation of new blood vessels, is important for normal development and the maintenance of tissue homeostasis. However, in many diseases, the events that are normally tightly controlled become disrupted, which can lead to aberrant and extensive growth of the blood vessels. Pathological angiogenesis is a key feature in many vision-threatening eye diseases and in cancers. Vascular endothelial growth factor (VEGF) is the main driver of pathological angiogenesis and VEGF- inhibitors are commonly used as a treatment for neovascular diseases. However, the therapies are not always sufficiently effective, and resistance to them can develop. Thus, more effective and specific therapeutics are needed. For this reason, the better understanding of the molecular mechanisms behind the pathologies is important. The main aim of this dissertation was to study the effect of different genes on the formation of new blood vessels and to identify new factors that contribute to the regulation of angiogenesis. The dissertation consists of four studies that addressed the role of angiogenesis mainly in eye diseases. These studies made use of experimental models of retinopathies as well as samples from human patients with proliferative diabetic retinopathy and retinal vein occlusion. The first study investigated the role of R-Ras in the regulation of angiogenesis in ischemic retinopathy. We found that R-Ras was important for the maintenance of vascular stability. The same ischemic retinopathy model was used in the second study, in which we showed that the function of myeloid-specific furin for retinal angiogenesis plays a role in retinal revascularization. In the third study we performed the most comprehensive proteomic profiling of the oxygen-induced retinopathy model to date. We identified novel proteins that may drive the pathogenesis and neovascularization in the mouse retinopathy model. These proteins may have relevance to human retinopathy as well as new potential drug targets. The fourth study explored the role of syndecan 4 (SDC4) in angiogenesis using several angiogenesis models. We demonstrated that SDC4 is needed for VEGF-\- driven pathological angiogenesis and VE-cadherin trafficking. The therapeutic potential of SDC4 inhibition was tested in an experimental model of wet age-related macular degeneration (AMD), and we found that it was as effective as a drug commonly used in the clinic for the treatment of wet AMD. The results of these studies improve the knowledge of the factors that affect angiogenesis. These results could be useful in the future development of new drugs for diseases that involve pathological angiogenesis