Patent Application: US-17515302-A

Abstract:
vegf - b is shown to be needed for cardiac muscle revascularization after heart infarction , and methods of promoting or stimulating vascular development , e . g . angiogenesis and / or arteriogenesis , particularly in ischemic mammals , are disclosed .

Description:
chronic myocardial ischemia is achieved by ligation of the left anterior descending ( lad ) coronary artery using 8 weeks old normal and vegf - b deficient mice after anaesthesia . the vegf - b deficient mice are described in published international application no . wo 98 / 36052 . prior to challenge , the capillary densities of both wild type ( normal ) and knock out ( vegf - b deficient ) were the same . seven days after lad ligation , infarcted hearts were fixed and collected . the infarcted hearts were sectioned longitudinally ( 6 m ). heamatoxylin & amp ; eosin and immunohistochemistry staining were performed using thrombomodulin for endothelial cells and smooth muscle alpha - actin for smooth muscle cells as markers . antibodies to thrombomodulin were obtained from dr . ed conway of leuven university in belgium . antibodies to smooth muscle cell alpha actin were obtained commercially ( dako , x0910 , denmark ). infarcted area and vessel densities were calculated using a quantinet q600 image analysis system ( leica , brussels , belgium ). data were analysed using the student t test . seven days after lad artery ligation , vessel densities in the infarcted area were calculated using thrombomodulin ( tm ) and smooth muscle cell alpha - actin ( smc ) as markers . the results are illustrated in fig1 ( a ) and ( b ) and 3 ( a ) and ( b ), respectively , and tabulated graphically in fig2 and 4 . [ 0062 ] fig1 ( a ) and ( b ) show thrombomodulin staining in wild type ( wt ) and vegf - b deficient ( ko ) hearts after infarction . the endothelium cell marker , thrombomodulin stains endothelium cells lining the lumen of vessels . in the infarcted area of the wild type heart ( wt ), there are more positive staining of different sized vessels compared with that of the vegf - b deficient one ( ko ). as can be seen from fig2 using thrombomodulin as the marker , total vessel density in the vegf - b deficient mice was about 63 % of that of the normal mice on average ( p & lt ; 0 . 01 , n = 9 ). when the vessels were classified into three different groups , large , medium - sized and small vessels , results showed the same deficiency in all the groups ( vegf - b deficient mice : 18 . 6 ± 3 . 1 large vessels / mm 2 , 34 . 8 ± 8 . 5 medium - sized vessels / mm 2 , 75 ± 20 . 3 small vessels / mm 2 ; normal mice : 30 . 6 ± 8 . 5 large vessels / mm 2 1 54 . 9 ± 13 . 4 medium - sized vessels / mm 2 , 117 . 7 ± 20 . 8 small vessels / mm 2 , n = 9 each group , p & lt ; 0 . 05 in all the groups ) fig3 ( a ) and ( b ) show the results of smooth muscle cell alpha actin staining in wild type ( wt ) and vegf - b deficient ( ko ) hearts after infarction . the smooth muscle cell marker , smooth muscle cell alpha actin , stains smooth muscle cells surrounding the vessels . in the infarcted area of the wild type heart ( wt ), there are more positive staining of different sized vessels compared with those in the vegf - b deficient heart ( ko ). as can be seen from the graph in fig4 when tissue sections were stained with smooth muscle alpha - actin for vascular smooth muscle cells , the vegf - b deficient mice showed the same revascularization defect after heart infarction observed in the previous test . the vegf - b deficient animals displayed only 61 % of the normal vessel density in total ( p & lt ; 0 . 05 , n = 9 ). vessels in different sizes showed the same result ( vegf - b deficient mice : 5 . 5 ± 1 . 3 large vessels / mm 2 , 8 . 0 ± 2 . 0 medium - sized vessels / mm 2 , 13 . 8 ± 8 . 3 small vessels / mm 2 ; normal mice : 7 . 7 ± 1 . 9 large vessels / mm 2 , 14 . 0 ± 4 . 8 medium - sized vessels / mm 2 , 22 . 7 ± 7 . 2 small vessels / mm 2 , n = 9 each group , p & lt ; 0 . 05 in all the groups ). thus , the test data clearly demonstrates that vegf - b deficiency leads to impaired revascularization after heart infarction . it follows that the presence of vegf - b is necessary for revascularization after heart infarction and that in cases of cardiac ischemia , administration of a therapeutically effective amount of vegf - b may stimulate or promote compensatory vascular development . the angiogenic activity of vegf - b is tested using the mouse cornea model and in the avian chorioallantoic membrane tests following the procedure described in cao et al ., proc . natl . acad . sci . usa 95 : 14389 - 14394 , 1998 . the ability of vegf - b to induce angiogenesis in ischemic tissue is tested following the procedure described in witzenbichler et al ., am . j . pathol . 153 : 381 - 394 , 1998 . the cdna encoding human vegf - b 167 was cloned between the strong enhancer / promoter of the cytomegalovirus ( cmv ) immediate early genes and the sv40 polyadenylation signal of the bacterial plasmid paccmvplpa ( gomez - foix a . et al . ( 1992 ) j . biol . chem . 267 , 25129 and janssens s . p . et al . ( 1996 ) j . clin . invest . 98 ( 2 ) 317 ). the plasmid also contained e1a - deleted sequences of type 5 adenovirus including the origin of replication , the packaging signal and a polylinker . recombinant adenovirus was generated through homologous recombination with pjm17 , a bacterial plasmid containing the full - length adenoviral genome , following cotransfection in e1a - transformed human embryonic retinal ( 911 ) cells . the presence of vegf - b cdna in virion dna isolated from infected 911 cells was confirmed by pcr analysis . viral isolates containing vegf - b ( adcmv . vegf - b ) were amplified on confluent 911 cells and , after appearance of cytopathic effects , isolated , precipitated , and concentrated by discontinuous cscl gradient . viral titers were determined by infection of monolayers of 911 cells with serial dilutions of the recombinant adenovirus . for in vivo studies , 3 × 10 9 plaque forming units ( pfu ) vegf - b or control rr5 adenovirus were injected intravenously per mouse . wild - type ( wt ) mice were subjected to ligation of the left descendens coronary artery as described above under example 1 to induce myocardial ischemia . thereafter , a group of eleven test animals were treated intravenously with 3 × 10 9 pfu adcmv . hvegf - b from example 4 per mouse . as a negative control , another group of 13 animals was treated with 3 × 10 9 pfu of adrr5 virus per mouse . revascularization of infarcts was measured by counting the number of vessels per mm 2 of infarct area within seven days after ligation of the coronary artery . results are shown in the following table 1 : the results clearly demonstrate that the gene therapy treatment with vegf - b dna in a viral vector resulted in increased re - vascularization of the infrarct area compared to the negative control . wild - type ( wt ) mice were subjected to ligation of the left descendens coronary artery as described above under example 1 to induce myocardial ischemia . a group of five ischemic test animals was thereafter treated with a solution containing recombinant human vegf - b 167 ( r vegf - b 167 ) supplied by amrad corporation limited of melbourne , australia . the solution containing recombinant human vegf - b was administered via an osmotic minipump implanted subcutaneously on the back of each mouse . the protein leaks out into the extracellular space and is resorbed in the blood . the recombinant vegf - b was administered at a rate of 10 micrograms of active substance per mouse per week . as a negative control a second group of five animals was treated with an identical amount of physiological saline solution . revascularization of infarcts was measured by counting the number of vessels per mm 2 of infarct area within seven days after ligation of the coronary artery . results are shown in the following table 2 : the foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting . since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art , the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof . 1 . carmeliet , p ., mechanisms of angiogenesis and arteriogenesis . nat med , 2000 . 6 ( 4 ): p . 389 - 95 . 2 . carmeliet , p . and r . k . jain , angiogenesis in cancer and other diseases . nature , 2000 . 407 ( 6801 ): p . 249 - 57 . 3 . li , x . and u . eriksson , novel vegf family members : vegf - b , vegf - c and vegf - d . int j biochem cell biol , 2001 . 33 ( 4 ): p . 421 - 6 . 4 . carmeliet p , et al ., abnormal blood vessel development and lethality in embryos lacking a single vegf allele . nature , 1996 . 380 ( 6573 ): p . 435 - 9 . 5 . ferrara n , et al ., heterozygous embryonic lethality induced by targeted inactivation of the vegf gene . nature , 1996 . 380 ( 6573 ): p . 439 - 42 . 6 . poltorak , z ., t . cohen , and g . neufeld , the vegf splice variants : properties , receptors , and usage for the treatment of ischemic diseases . herz , 2000 . 25 ( 2 ): p . 126 - 9 . 7 . carmeliet , p ., vegf gene therapy : stimulating angiogenesis or angioma - genesis ? 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