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:
STATEMENT OF U.S. GOVERNMENT INTEREST 
       [0001]    This invention was made with U.S. government support under grant number R01 HL 110737-01 awarded by the National Institutes of Health, Heart, Lung and Blood Institute. The U.S. government has certain rights in the invention. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present disclosure is directed to materials and methods for treating endothelial dysfunction in a human subject and for monitoring efficacy of mesenchymal stem cell therapy in a subject suffering from a cardiovascular disorder. 
       BACKGROUND 
       [0003]    Heart failure (HF) remains a leading cause of morbidity and mortality in the United States 1 . The failing circulation is characterized not only by depressed cardiac function, but also by endothelial dysfunction 2 . Endothelial dysfunction produces increased systemic vascular resistance, which augments stress on the failing heart, and contributes to HF symptomology 3,4 . Endothelial dysfunction is also a crucial component of the pathophysiology of numerous cardiovascular (CV) disorders and manifests in patients with CV risk factors such as atherosclerosis, hypertension, and diabetes mellitus 5,6 . Moreover, endothelial progenitor cells (EPCs) regulate the health of the vasculature by incorporating into the endothelium, replacing injured endothelial cells, and secreting angiogenic factors that activate mature endothelial cells 7,8 . Notably, HF patients have decreased circulating EPC levels and bioactivity 7 . 
       SUMMARY 
       [0004]    A method of monitoring efficacy of a therapy for endothelial dysfunction is provided. In one aspect, the method comprises determining levels of Vascular Endothelial Growth Factor (VEGF) and endothelial progenitor cells (EPCs) in a first sample from the subject before administration of a therapy for endothelial dysfunction; and determining levels of VEGF and EPCs in a second sample from the subject after administration of the therapy for endothelial dysfunction; wherein a decreased level of VEGF and an increased level of EPCs in the second sample compared to levels of VEGF and EPCs in the first sample is indicative of effective treatment of the endothelial dysfunction. In some embodiments, the therapy for endothelial dysfunction comprises mesenchymal stem cell therapy. In some embodiments, the mesenchymal stem cell therapy comprises the administration of allogeneic mesenchymal stem cells to the subject. 
         [0005]    Alternatively, the invention provides a method of monitoring efficacy of a therapy for endothelial dysfunction comprising determining levels of Vascular Endothelial Growth Factor (VEGF) and endothelial progenitor cells (EPCs) in a sample from the subject after administration of the therapy for endothelial dysfunction, and comparing the levels of VEGF and EPC to a predetermined criterion, e.g., levels of VEGF and EPC from healthy subjects. A decreased level of VEGF (such that the level of VEGF in the sample is commensurate with a level of VEGF in healthy subjects) and an increased level of EPCs (such that the level of EPCs is commensurate with a level of EPCs in healthy subjects) in the sample compared to the predetermined criterion is indicative of effective treatment of the endothelial dysfunction. Any therapy for endothelial cell dysfunction is contemplated herein; in one aspect, the therapy is a cell-based therapy, such as administration of stem cells (e.g., mesenchymal stem cells). 
         [0006]    Also described herein is a method of monitoring efficacy of a mesenchymal stem cell therapy in a subject suffering from a cardiovascular disorder comprising determining a level of Vascular Endothelial Growth Factor (VEGF) in a sample from the subject after administration of the stem cell therapy, and comparing the level of VEGF to a predetermined criterion, e.g., level of VEGF from healthy subjects. A decreased level of VEGF in the sample such that it is commensurate with a level of VEGF in healthy subjects is indicative of effective stem cell therapy. 
         [0007]    The steps of determining the level of VEGF and/or EPCs in the sample is performed by any means, such as those known in the art and described below. For example, the determining the level of VEGF in a sample entails quantifying the amount of VEGF protein in the sample in some aspects of the invention, although quantifying the amount of VEGF DNA or mRNA also is contemplated. In some embodiments, the method comprises isolating EPCs from the sample, culturing the EPCs for at least 24 hours, and determining the number of colony forming units (CFUs). 
         [0008]    In some embodiments, the sample is a blood sample or serum sample. 
         [0009]    The disclosure further provides a method of identifying endothelial dysfunction in a subject comprising determining a level of Vascular Endothelial Growth Factor (VEGF) in a sample from the subject; and determining a level of endothelial progenitor cells (EPCs) in the sample from the subject, wherein an elevated level of VEGF in the sample and a decreased level of EPC in the sample is indicative of endothelial dysfunction in the subject 
         [0010]    In another aspect, described herein is a method of treating endothelial dysfunction in a subject in need thereof comprising administering to a subject allogeneic mesenchymal stem cells (MSCs) in an amount effect to decrease circulating levels of VEGF and increase levels of EPCs in the subject, thereby treating the endothelial dysfunction. In some embodiments, the subject is suffering from a cardiovascular disorder such as, for example, heart failure, idiopathic dilated cardiomyopathy and ischemic cardiomyopathy. In various embodiments, the method improves vascular tone. 
         [0011]    The mesenchymal stem cells are administered according to any suitable method, including those known in the art. In some embodiments, the cells are administered locally. In some embodiments, the cells are administered by transendocardial injection. 
         [0012]    The mesenchymal stem cells are optionally administered in an amount ranging from about 20 million to about 100 million cells. In some embodiments, the mesenchymal stem cells are administered in an amount ranging from about 20 million to about 30 million cells, or about 20 millions to about 40 million cells, or about 20 million to about 50 million cells, or about 20 million to about 60 million cells, or about 20 million to about 70 million cells, or about 20 million to about 80 million cells, or about 20 million to about 90 million cells, or about 30 million to about 50 million cells, or about 30 million to about 70 million, or about 30 million to about 90 million cells, or about 50 million to about 100 million cells. In some embodiments, about 20 million, or about 25 million, or about 30 million, or about 35 million, or about 40 million, or about 45 million, or about 50 million, or about 55 million, or about 50 million, or about 55 million, or about 60 million, or about 65 million, or about 70 million, or about 75 million, or about 80 million, or about 85 million, or about 90 million, or about 95 million, or about 100 million mesenchymal stem cells are administered to the subject. 
         [0013]    In some embodiments, administration of the mesenchymal stem cells to the subject results in an increase in flow-mediated vasodilation (FMD) in the subject of at least 3%. 
         [0014]    In any of the methods described herein, the subject is a human subject. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0015]      FIG. 1 . Endothelial function in patients with heart failure, including dilated and ischemic cardiomyopathy.  FIG. 1A  shows that patients (n=22) in the study had impaired EPC-CFUs compared to healthy controls (n=10, ****P&lt;0.001).  FIG. 1B  shows that patients (n=22) in the study had reduced FMD % compared to healthy controls (n=10, **P=0.0017). 
           [0016]      FIG. 2 . Endothelial colony forming units in heart failure patients treated with either allogeneic or autologous mesenchymal stem cells (MSCs).  FIG. 2A  shows that patients treated with allogeneic MSCs had a significant improvement in EPC-CFUs 3 months post treatment (n=15, ****P&lt;0.001).  FIG. 2B  shows that patients treated with autologous MSCs had no change in EPC-CFUs post treatment (n=7).  FIGS. 2C and 2E  show representative EPC-CFUs plated on fibronectin for 5 days before MSC administration (magnification 20×).  FIGS. 2D and 2F  shows representative EPC-CFYs plated on fibronectin for 5 days after MSC administration (magnification 20×). 
           [0017]      FIG. 3 . Flow mediated vasodilation (FMD) measurements before and after mesenchymal stem cell (MSC) treatment.  FIG. 3A  shows that patients treated with allogeneic MSCs had an increase in FMD % 3 months post injection (n=15, ***P&lt;0.001).  FIG. 3B  shows that patients treated with autologous MSCs had no significant difference in FMD % 3 months post injection (n=7, P=NS).  FIG. 3C  shows that correlation between the absolute change in FMD % and the absolute change in EPF-CFUs from baseline to 3 months post MSC injection in all patients (P&lt;0.001, R=0.684). 
           [0018]      FIG. 4 . Serum VEGF concentration in patients and controls.  FIG. 4A  demonstrates that patients (n=14) had a higher level of circulating VEGF compared to controls (n=9) at baseline (P=0.0009).  FIG. 4B  demonstrates that patients who received allogeneic MSCs (n=9) had a decrease in VEGF serum levels post injection (Δ−547.5±350.8 pg/mL), while patients who received autologous MSCs (n=5) had an increase in serum VEGF post injection (Δ4814.1±875.8), and there was a difference between groups (**P=0.0012).  FIG. 4C  shows that there is a correlation between EPC-CFUs and serum VEGF in patients at both baseline and 3 months post MSC treatment (R=−0.421, P=0.026).  FIG. 4D  shows that the change in EPC-CFUs from baseline to 3 months post treatment strongly correlated with the change in VEGF (R=−0.863, P&lt;0.001). 
       
    
    
     DETAILED DESCRIPTION 
       [0019]    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. 
         [0020]    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. 
         [0021]    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. 
         [0022]    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 . 
       EXAMPLES 
     Methods 
       [0023]    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. 
         [0024]    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. 
         [0025]    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. 
         [0026]    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). 
         [0027]    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. 
         [0028]    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. 
       Results 
       [0029]    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. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Summary of patients (n = 22) and healthy controls (n = 10). 
               
               
                 Patients are broken down by etiology and cell treatment: DCM patients 
               
               
                 receiving allogeneic MSCs (n = 9), DCM patients receiving autologous 
               
               
                 MSCs (n = 7) and ICM patients receiving allogeneic MSCs (n = 6).. 
               
             
          
           
               
                   
                 DCM 
                 DCM 
                 ICM 
                 Healthy 
               
               
                   
                 Allogeneic 
                 Autologous 
                 Allogeneic 
                 Controls 
               
               
                 Characteristic 
                 (N = 9, 27%) 
                 (N = 7, 21%) 
                 (N = 6, 18%) 
                 (N = 10, 33%) 
               
               
                   
               
               
                 Age - yr. 
                   
                   
                   
                   
               
               
                 Median 
                 60  
                 55 
                 71 
                 44 
               
               
                 Range 
                 45-70 
                 48-73 
                 65-75 
                 22-58 
               
               
                 Male sex- no. 
                 8 (89%) 
                 6 (86%) 
                 6 (100%) 
                  7 (70%) 
               
               
                 Race/Ethnicity 
               
               
                 White 
                 8 (89%) 
                 3 (43%) 
                 6 (100%) 
                  7 (70%) 
               
               
                 White/Hispanic 
                 0 (0%)  
                 4 (57%) 
                 0 (0%)  
                  3 (30%) 
               
               
                 Black 
                 1 (11%) 
                 0 (0%)  
                 0 (0%)  
                 0 (0%) 
               
               
                 History of CVD 
               
               
                 HF 
                 2 (22%) 
                 0 (0%)  
                 1 (17%)  
                 0 (0%) 
               
               
                 CAD 
                 1 (11%) 
                 0 (0%)  
                 6 (100%) 
                 0 (0%) 
               
               
                 CHF 
                 0 
                 1 (14%) 
                 4 (67%)  
                 0 (0%) 
               
               
                 History of Smoking 
                 4 (44%) 
                 4 (57%) 
                 4 (67%)  
                 0 (0%) 
               
               
                 Blood pressure- 
               
               
                 mmHg 
               
               
                 (systolic/diastolic) 
               
               
                 Median 
                 126/77 
                 108/65 
                 113/70 
                 118/67 
               
               
                 Range 
                 108-   
                 99-142/59-84 
                 85-134/53-  
                 111-130/59-   
               
               
                 Cholesterol 
               
               
                 Median 
                 202  
                 175  
                 147  
                 N/A 
               
               
                 Range 
                 129-282 
                 112-207 
                 129-178 
                 N/A 
               
               
                 C-Reactive Protein 
               
               
                 (mg/mL) 
               
               
                 Median 
                   0.3 
                   0.3 
                    0.15 
                 N/A 
               
               
                 Range 
                 0.2-0.4 
                 0.2-0.5 
                 0.1-0.6 
                 N/A 
               
               
                 Medications 
               
               
                 ASA/NSAIDS 
                 5 (56%) 
                 4 (57%) 
                 5 (83%)  
                 0 (0%) 
               
               
                 Beta-Blockers 
                 7 (78%) 
                  7 (100%) 
                 6 (100%) 
                 0 (0%) 
               
               
                 ACE 
                 6 (67%) 
                  7 (100%) 
                 6 (100%) 
                  1 (10%) 
               
               
                 Diuretics 
                 8 (89%) 
                 6 (86%) 
                 4 (67%)  
                 0 (0%) 
               
               
                 Statins 
                 3 (33%) 
                 3 (43%) 
                 6 (100%) 
                  1 (10%) 
               
               
                 Antiplatelet 
                 4 (44%) 
                 2 (29%) 
                 3 (50%)  
                 0 (0%) 
               
               
                 Other 
                  9 (100%) 
                  7 (100%) 
                 6 (100%) 
                 0 (0%) 
               
               
                   
               
             
          
         
       
     
         [0030]    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 ( FIG. 1 ). 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). 
         [0031]    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,  FIG. 2A ). On the other hand, patients who received autologous MSCs showed no improvement (Δ1±3, P=NS,  FIG. 2B ). Moreover, we compared allogeneic MSC treatment versus autologous MSC treatment, and allogeneic MSCs were superior in stimulating EPC colony formation (P=0.02). 
         [0032]    EPC-CFUs were also examined for morphology. EPCs from HF patients had disorganized and incomplete colony formation ( FIG. 2C  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 ( FIG. 2D  and F). These findings suggest that transendocardial MSC therapy stimulates EPC bioactivity in patients with HF of both ischemic and non-ischemic etiology. 
         [0033]    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,  FIG. 3A ). In contrast, patients who received autologous MSCs had no improvement and the majority of patients worsened 3 months post-treatment (Δ−0.46±3%,  FIG. 3B ). 
         [0034]    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,  FIG. 3C ). 
         [0035]    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,  FIG. 4A ). 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,  FIG. 4B ). Furthermore, there was a significant difference between patients who received allogeneic MSCs versus patients who received autologous MSCs (P=0.0012,  FIG. 4B ). 
         [0036]    VEGF levels correlated with EPC-CFUs (P=0.026, R=-0.421,  FIG. 4C ). 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,  FIG. 4G ). 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. 
       Summary 
       [0037]    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. 
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