Patent Application: US-201515527270-A

Abstract:
the present disclosure is directed to materials and methods for treating endothelial dysfunction in a subject in need thereof . methods of determining efficacy of treatment are also provided .

Description:
the present disclosure is based , at least in part , on the discovery that increased circulating levels of vascular endothelial growth factor ( vegf ), optionally in combination with decreased levels of endothelial progenitor cells ( epcs ), is indicative of endothelial dysfunction in a subject , and reduced levels of vegf and increased levels of epcs are indicative of effectiveness of therapy for endothelial dysfunction , vascular tone , and cardiovascular disease . data provided herein demonstrated that the administration of allogeneic mesenchymal stem cells ( mscs ) to subjects suffering from heart failure stimulated epc bioactivity and restored flow - mediated vasodilation towards normal . the ability of allogeneic mscs greatly exceeded that of autologous mscs in restoring endothelial function , enhancing epc cfu formation , and suppressing vegf levels . together these findings offer major new clinical insights into the bioactivity of mscs , suggest a novel therapeutic principle for disorders characterized by endothelial dysfunction , and provide useful diagnostic markers for detecting and measuring successful treatment . mscs are adult stem cells that are prototypically found in bone marrow and have the capacity to differentiate into multiple cell types . they stimulate the proliferation and differentiation of endogenous precursor cells and play a crucial role in maintaining stem cell niches 27 . in addition , mscs secrete paracrine factors which participate in angiogenesis , cardiomyogenesis , neovascularization , stimulation of other endogenous stem cells , and regulation of the immune system 28 , 29 . while mscs are known to stimulate cardiac precursor cells and cell cycle activity in the heart , their role in stimulating other endogenous precursor populations has heretofore been unknown . the data provided herein establish that mscs stimulate endogenous epc activation , increasing the number and quality of functional epcs . these findings suggest that augmentation of epcs may represent a novel mechanism of action by which mscs exert favorable biological effects . over the last decade , there has been an emerging interest in the use of mscs in cv disorders 30 . clinical trials have demonstrated a major safety profile for msc administration , and suggested efficacy in patients with hf 30 - 32 ; however , underlying mechanism ( s ) of action continue to be vigorously debated . the instant finding that allogeneic msc injections in patients with both ischemic and non - ischemic hf results in an improvement in endothelial function specifically by restoring epc function , fmd , and reducing vegf levels towards normal offers a major new insight into the mechanisms of action of mscs . in the study population , increased serum vegf correlated with diminished epc - cfus , consistent with the idea that vegf plays a compensatory role , a finding also reported in patients with cerebral aneurysm 26 . the findings provided herein establish that allogeneic mscs can be employed to stimulate epc bioactivity , improve arterial physiologic vasodilatory responses , and decrease unfavorable cytokine mobilization in patients with cv disease and other disorders associated with endothelial dysfunction . the data provided herein demonstrate that allogeneic mscs restored endothelial function in patients to a degree greatly exceeding that of autologous mscs . in the study described below , all allogeneic stem cell donors were healthy , young donors between the ages of 20 and 35 . patients receiving their own stem cells not only had underlying chronic diseases , but were also older ( between the ages of 45 and 75 ). autologous mscs aging may impair the survival , differentiation , and ability to recruit epcs to areas of damage , ultimately reducing their therapeutic efficacy 38 , 39 . study population : the hf patients were enrolled in poseidon - dcm ( nct01392625 ), “ a phase i / ii , randomized pilot study of the comparative safety and efficacy of transendocardial injection of autologous mesenchymal stem cells versus allogeneic mesenchymal stem cells in patients with nonischemic dilated cardiomyopathy ” and in trident ( nct02013674 ), “ the transendocardial stem cell injection delivery effects on neomyogenesis study ”. in poseidon - dcm , patients were randomized to receive by transendocardial delivery either 100 million autologous or allogeneic mscs . autologous mscs were derived from the patient &# 39 ; s bone marrow ( iliac crest aspiration ) 4 - 6 weeks before cardiac catheterization . in the trident study , icm patients were randomized to receive either 20 or 100 million allogeneic mscs transendocardially . allogeneic mscs were manufactured by the university of miami cell manufacturing program . healthy subjects ( n = 10 ) were enrolled ranging in ages from 22 - 58 years and both genders . all subjects provided written informed consent and the study was approved by the university of miami institutional review board . endothelial colony forming units ( epc - cfus ): peripheral blood samples were obtained from patients before and 3 months after msc injection . epcs were isolated from samples using ficoll - paque and 5 million cells were seeded on 6 - well fibronectin - coated dishes ( bd biosciences ) in cfu - hill medium ( stem cell technologies , cat # 05900 ) 17 . the non - adherent cells were collected 48 hours later and 1 million cells were seeded on 24 - well fibronectin - coated dishes . on day 5 , epc - cfus were counted in 5 wells and the average was obtained . flow - mediated vasodilation ( fmd ): brachial artery diameter measurements and fmd % were performed in the morning , after an overnight fast . the subjects &# 39 ; right arm was immobilized in an extended position , and the brachial artery was scanned via ultrasound 5 - 10 cm above the antecubital fossa l7 , 18 . a brachial cuff was then inflated to a supra - systolic pressure ( 40 to 50 mmhg above systolic pressure ) for 5 minutes . subsequently , the cuff was deflated and the brachial artery diameter was recorded for 3 minutes . vegf elisa : serum vascular endothelium growth factor ( vegf ) levels ( invitrogen # khg0111 ) were measured in dcm patients at baseline and 6 months after allogeneic ( n = 6 ) or autologous ( n = 5 ) msc treatment . in icm patients ( n = 4 ), vegf was measured at baseline and 3 months after allogeneic msc treatment . dcm and icm patients who received allogeneic mscs were combined and compared to patients who received autologous . lastly , vegf was measured in controls ( n = 9 ). c - reactive protein : c - reactive protein ( crp ) was evaluated using a nephelometric method in which blood plasma samples were incubated with latex particles coated with crp monoclonal antibodies . the analytical sensitivity was 0 . 2 mg / l and results were reported in mg / l . statistical analysis : to assess the difference between autologous and allogeneic groups , an unpaired , two - tailed t - test was used . to measure the difference before and after treatment in each group , both a paired , two - tailed t - test and a one - way anova was utilized . correlations were measured using pearson correlation , assuming a gaussian distribution . data are presented as mean and standard deviation of the mean . to determine the difference between huvec treatment groups , an unpaired , two - tailed t - test was used . baseline characteristics : a total of 22 patients were analyzed for this study . fifteen patients received allogeneic mscs and 7 patients received autologous mscs . baseline characteristics of the study subjects are summarized in table 1 . age and sex were evenly distributed . epc - cfus and fmd in heart failure patients and healthy subjects : patients with ischemic ( n = 15 ) as well as non - ischemic ( n = 6 ) cardiomyopathy had endothelial dysfunction at baseline , characterized by a reduced ability to form epc - cfus and an impaired fmd response ( fig1 ). specifically , patients had decreased epc - cfu counts compared to healthy controls ( 4 ± 3 vs . 25 ± 16 , respectively , p & lt ; 0 . 001 ) as well as diminished fmd % ( 5 . 6 ± 3 . 2 vs . 9 . 2 ± 4 , respectively , p = 0 . 0017 ). epc - cfus in heart failure patients treated with mscs : patients were assessed for epc - cfus before and 3 months after msc treatment . patients who received allogeneic mscs had a significant improvement in the number of epc - cfus post - treatment ( δ10 ± 5 , p & lt ; 0 . 001 , fig2 a ). on the other hand , patients who received autologous mscs showed no improvement ( δ1 ± 3 , p = ns , fig2 b ). moreover , we compared allogeneic msc treatment versus autologous msc treatment , and allogeneic mscs were superior in stimulating epc colony formation ( p = 0 . 02 ). epc - cfus were also examined for morphology . epcs from hf patients had disorganized and incomplete colony formation ( fig2 c and e ), resulting in clusters that failed to form functional colonies . three months after allogeneic msc treatment , patient colonies were organized and healthy in appearance ( fig2 d and f ). these findings suggest that transendocardial msc therapy stimulates epc bioactivity in patients with hf of both ischemic and non - ischemic etiology . fmd in heart failure patients treated with mscs : all patients were evaluated using brachial artery fmd before and 3 months after msc injection . patients who received allogeneic mscs had a dramatic improvement in fmd % ( δ3 . 7 ± 3 %, p = 0 . 001 , fig3 a ). in contrast , patients who received autologous mscs had no improvement and the majority of patients worsened 3 months post - treatment ( δ − 0 . 46 ± 3 %, fig3 b ). the difference between treatment with autologous mscs and allogeneic mscs was analyzed . there was a striking difference between the two cell types ( p = 0 . 005 ), suggesting that autologous mscs do not restore endothelial function in this patient population . we also assessed the correlation between epc - cfus and fmd % in all patients and found a highly significant correlation between δfmd % and δepc - cfus ( p & lt ; 0 . 001 , r = 0 . 684 , fig3 c ). assessment of vegf in patients receiving autologous and allogeneic mscs : circulating vegf levels in patients and healthy control serum was determined . at baseline , patients had profoundly elevated circulating vegf compared to controls ( 1130 . 3 ± 803 . 3 vs . 2 . 0 ± 5 . 9 pg / ml , p & lt ; 0 . 001 , fig4 a ). allogeneic mscs reduced vegf levels (− 547 . 5 ± 350 . 8 pg / ml , p = 0 . 002 ), while patients who received autologous mscs had an increase in vegf ( 814 . 1 ± 875 . 8 pg / ml , fig4 b ). furthermore , there was a significant difference between patients who received allogeneic mscs versus patients who received autologous mscs ( p = 0 . 0012 , fig4 b ). vegf levels correlated with epc - cfus ( p = 0 . 026 , r =- 0 . 421 , fig4 c ). even more striking , there was a significant correlation between the change in vegf and the change in epc - cfus from baseline to three months post treatment ( r = 0 . 863 , p & lt ; 0 . 001 , fig4 g ). notably , high levels of vegf correlated with low levels of epcs , evidenced by the autologous group . conversely , lower levels of vegf correlated with high levels of epc - cfus , illustrated by the allogeneic group . taken together , these data demonstrate mscs stimulate epc mobilization and suppress compensatory elevations in circulating vegf concentrations . this example demonstrates a potent and clinically relevant efficacy outcome of transendocardial therapy with mscs in patients with advanced hf . allogeneic mscs restored flow mediated brachial artery dilation , epc bioactivity , and vegf levels towards normal . as abnormalities in the vascular function of patients with cv disease is shown to be highly predictive of adverse outcomes and disease progression , targeting endothelial function is a significant therapeutic strategy . the data also reveals vegf as a novel indicator for successful treatment of endothelial dysfunction , restoration of vascular tone , and alleviation of complications of cardiovascular disease . subjects demonstrating improved fmd displayed reduced circulating vegf levels , as well as higher levels of epc - cfus , compared to heart failure subjects demonstrating no improvement in fmd . 1 . go a s , mozaffarian d , roger v l , et al . heart disease and stroke statistics — 2014 update : a report from the american heart association . circulation 2014 ; 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