Targeting Neuropilin-1 to Inhibit VEGF Signaling in Cancer: Comparison of p Therapeutic Approaches Feilim Mac Gabhann* , Aleksander S. Popel Department of Biomedical Engineering Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America States of America
B Background k d � Angiogenesis( neovascularization ): the growth of new blood vessels from pre- existing microvessels . existing microvessels � physiological & pathological conditions including cancer, cardiovascular conditions including cancer, cardiovascular disease and wound healing VEGF is a critical regulator of angiogenesis
VEGF i VEGF signaling pathway li th F Family of secreted glycoproteins and critical regulators of il f t d l t i d iti l l t f � angiogenesis VEGFs; � In vitro � endothelial cell survival proliferation and migration In vitro � endothelial cell survival, proliferation and migration. In vivo � vascular permeability, activation of endothelial cells. VEGF121 VEGF165 VEGF121, VEGF165 � � VEGFR1,VEGFR2 (signaling VEGF receptor tyrosine � kinases) Neuropilin-1 (non-signaling co-receptor) Neuropilin 1 (non signaling co receptor) � �
VEGF121 i VEGF121 isomer � Only direct binding to VEGFR1 and VEGFR2 Only direct binding to VEGFR1 and VEGFR2 � No binding to free Neuropilin-1 (but can bind to VEGFR1 bound Neuropilin-1)
VEGF165 i VEGF165 isomer � Direct binding to VEGFR1 and VEGFR2 Di t bi di t VEGFR1 d VEGFR2 � Interaction with Neuropilin-1 and proteoglycans of the extracellular matrix. * GAG represents ECM bi di ECM binding sites
VEGF165 i VEGF165 isomer (continues) ( ti ) Two ways of binding to VEGFR2: � Directly Two ways of binding to VEGFR2: � Directly � Binding to Neuropilin-1 that inturn induces VEGF165-VEGFR binding
Why is Neuropilin-1 targeted in therapeutic approaches? !!! !!! VEGF165 is the only identified VEGF isomer which induces pathological angiogenesis. Since Neuropilin directly interacts and has an influence in direction of its binding to VEGFRs, Thus usable for inhibiting VEGF signaling
P Previously, i l Authors developed computational models of VEGF Authors developed computational models of VEGF � � interactions with endothelial cell receptors in vitro (by using published experimental data to estimate the kinetic rate of VEGFR2-Neuropilin coupling with VEGF165). As a result, VEGFR2-Neuropilin coupling is sufficient to account for the observed differential effects of VEGF isoforms on multiple cell types (several cell types were used in model). � In this article, extension of validated models in order to predict in vivo behaviour of the system by including ECM and b basement membranes as well as multiple cell types t b ll lti l ll t (tumor & endothelial) and geometrical parameters characteristic of this tissue.
Methods (summary) ) M th d (
M th d Methods -1- 1 Three methods for targeting Neuropilin-VEGF165 Th th d f t ti N ili VEGF165 � interaction 1 ) 1-) Blocking Neuropilin expression, l k l by using siRNA or other methods to prevent the synthesis of the protein in the cells in the cells.
R Results -1- lt 1 Decrease in the Neuropilin expression � � transient decrease in VEGF-VEGFR2 formation (why transient?) Decrease in binding VEGF165 to VEGFR2 Less VEGF165 bound= Less VEGF165 internalized Increase in unbound VEGF165 I i b d VEGF165 Increase in binding to VEGFR2 and its signaling (VEGF121-VEGFR2 formation is not affected � from these treatments) * * in vivo endothelial cell type * * in vivo, endothelial cell type
M th d Methods -2- 2 2-) Blocking Neuropilin – VEGF165 interaction, by using a second protein that occupies VEGF165 binding site of Neuropilin-1 and thus inhibits its binding and thus inhibits its binding (for example a fragment of Placental Growth Factor- PIGF2-)
R Results -2- lt 2 Bl Blockage of VEGF165-Neuropilin interaction k f VEGF165 N ili i t ti results in a transient decrease in VEGF165- VEGFR2 formation (why transient?) Neuropilin induced VEGF165-VEGFR2 coupling decreased More unbound VEGF165 in the interstitial More unbound VEGF165 in the interstitial space = less internalized VEGF165 molecules Loss of Loss of Also constant Also constant inhibition inhibiton cannot because of obtained because of more VEGF165 internalization of concentration inhibitors after some time
M th d Methods -3- 3 3 ) Bl 3-) Blocking Neuropilin –VEGFR2 ki N ili VEGFR2 coupling, by using an antibody that competes with VEGFRs but not VEGF165 binding
R Results -3- lt 3 Thi bl This blocking results in significant ki lt i i ifi t � inhibition in VEGF165-VEGFR2 coupling (NOT transient!!-how?) VEGF165 can bind Neuropilin-1 but not VEGF165 can bind Neuropilin 1 but not VEGFR2 (because Neuropilin cannot bind VEGFR2) Less amount of VEGF165 binding= less amount of VEGF165 internalization= more concentrated VEGF165 in the interstitial space (Since the concentration interstitial space (Since the concentration of inhibitor is stable, it cannot prevent inhibitors’ effect by increased concentration) * * After 48 hours, inhibition is sustained, inhibitor is internalized
R Results (summary) lt ( ) In the first two methods, inhibiton of VEGF165-VEGFR2 I th fi t t th d i hibit f VEGF165 VEGFR2 � formation is less than 30% In the last method it is more than 80% (maximal average � of inhibition over 48 hours) of inhibition over 48 hours) Although blocking VEGFR2-Neuropilin coupling is more � effective, this does not mean this method is useful in , inhibition of angiogenesis and vascular growth. Other cytokines and VEGF signaling is important!! � High VEGFR1 expression: increase VEGF165-VEGFR1 � binding due to the decrease in expression or occupation of bi di d h d i i i f Neuropilin. � Tum or specificity: expression levels of receptors � dependent to tumor types Tumors which express low- dependent to tumor types. Tumors which express low- level of Neuropilin or high-level of VEGFR1 are less effected from these treatments.
Di Discussion i Effi Efficacy depends on d d Tumor type = expression levels of receptors and ligands � Timing (48 hours) � Concentration of inhibitors in the interstitial space C t ti f i hibit i th i t titi l � � Does not include pharmacokinetics, just computational model of VEGF pathway in vivo model of VEGF pathway in vivo. � The proteins that are used in this model are not the only proteins that inhibit Neuropilin, others may be designed or discovered with similar properties in order to increase discovered with similar properties in order to increase efficiency.
Questions? Questions? Thanks! Thanks!
Recommend
More recommend
