Materials and methods for monitoring vascular endothelial function

Claim 2

1 . A method for monitoring vascular endothelial function in a patient, comprising: (a) obtaining a test blood specimen from the patient; (b) measuring the amount of endothelial cells within the test blood specimen, wherein the endothelial cells are selected from the group consisting of circulating endothelial cells, endothelial progenitor cells, or both circulating endothelial cells and endothelial progenitor cells; and (c) determining the activation status of the endothelial cells within the test blood specimen.

Claim 3

2 . The method of claim 1 , further comprising: (d) correlating the amount of endothelial cells and their activation status with one or more risk factors for disease.

Claim 4

3 . The method of claim 1 , wherein the risk factors associated with disease are selected from the group consisting of living habits, atherogenic traits, and innate susceptibility.

Claim 5

4 . The method of claim 3 , wherein the risk factors associated with disease are selected from the group consisting of overeating of saturated fats; high cholesterol; high caloric intake; physical indolence; cigarette habit; type A behavior; unrestrained weight gain; elevated fibrinogen; leukocytosis; hypertension; genetic predisposition to a disorder selected from the group consisting of dyslipidemia, hypertension, and diabetes; and strong family history of premature cardiovascular disease.

Claim 6

5 . The method of claim 1 , further comprising: (d) determining whether the patient is at risk for a disease based upon the activation status of the endothelial cells within the test blood specimen.

Claim 7

6 . The method of claim 5 , wherein the disease is a vascular disease condition.

Claim 8

7 . The method of claim 6 , wherein the vascular disease condition is selected from the group consisting of atherosclerosis, hypertension, ischemia, ischemia reperfusion injury, heart failure, arrhythmias, cardiotoxicity, hypertrophy, stroke, deep brain thrombosis, intravascular coagulopathy, Kasabach-Merritt syndrome, pulmonary embolism, myocardial infarction, and thromboembolic complication of surgery.

Claim 9

8 . The method of claim 1 , wherein the activation status of the endothelial cells is determined by measuring at least one of the endothelial activation markers selected from the group consisting of surface adhesion molecules, procoagulant factors, endothelins, growth factor receptors, nitric oxide, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), prostaglandins I2 (PGI2), tissue factors, heme oxygenase (HO), tissue plasminogen activator (tPA), mitochondria superoxide dismutase (MnSOD), Cu/Zn superoxide dismutase (Cu/Zn SOD), tumor growth factor-beta (TGF-β), cyclooxygenase 1 (COX-1), cyclooxygenase-2 (COX-2), vascular cell adhesion molecule (VCAM), vascular endothelial growth factor (VEGF), VEGF receptor, intercellular adhesion molecule (ICAM), E-selectin, and P-selectin.

Claim 10

9 . The method of claim 1 , wherein the activation status of the endothelial cells is determined by measuring at least one of the endothelial activation markers selected from the group consisting of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule (VCAM), and heme oxygenase (HO).

Claim 11

10 . The method of claim 1 , wherein the endothelial cell activation marker is a vasoactive compound selected from the group consisting of endothelins, nitric oxide, and prostaglandins.

Claim 12

11 . The method of claim 1 , wherein the endothelial cell activation marker is VCAM-1.

Claim 13

12 . The method of claim 1 , wherein the endothelial cell activation marker is ICAM-1.

Claim 14

13 . The method of claim 1 , wherein the endothelial cell activation marker is E-selectin.

Claim 15

14 . The method of claim 1 , wherein the endothelial cell activation marker is VEGF receptor.

Claim 16

15 . The method of claim 1 , wherein the amount of endothelial cells within the test blood specimen is measured by flow cytometry.

Claim 17

16 . The method of claim 1 , wherein at least one of the endothelial cell activation markers is measured by an immunoassay.

Claim 18

17 . The method of claim 1 , wherein at least one of the endothelial cell activation markers is an enzyme, and wherein the activation status of the endothelial cells is determined by measuring the enzymatic activity exhibited by the enzyme.

Claim 19

18 . The method of claim 17 , wherein the enzyme is selected from the group consisting of endothelial nitric oxide synthase, inducible nitric oxide synthase, and heme oxygenase.

Claim 20

19 . The method of claim 1 , wherein at least one of the cardothelial cell activation markers is an enzyme, and wherein the activation status of the endothelial cells is determined by measuring the amount of the enzyme, measuring the amount of RNA encoding the enzyme, and measuring the amount of enzymatic activity exhibited by the enzyme.

Claim 21

20 . A method of diagnosing or monitoring a vascular disease in a patient, said method comprising: (a) administering a therapeutic vascular agent to the patient; (b) obtaining a test blood specimen from the patient; (c) measuring the amount of vascular agent within the blood specimen; (d) measuring the amount of endothelial cells within the test blood specimen, wherein the endothelial cells are selected from the group consisting of circulating endothelial cells, endothelial progenitor cells, and both circulating endothelial cells and endothelial progenitor cells; (e) determining the activation status of the endothelial cells within the test blood specimen; and (f) correlating the amount of circulating cells and their activation status with the amount of vascular agent within the blood specimen.

Claim 22

20 . A method of diagnosing or monitoring a vascular disease in a patient, said method comprising: (a) administering a therapeutic vascular agent to the patient; (b) obtaining a test blood specimen from the patient; (c) measuring the amount of vascular agent within the blood specimen; (d) measuring the amount of endothelial cells within the test blood specimen, wherein the endothelial cells are selected from the group consisting of circulating endothelial cells, endothelial progenitor cells, and both circulating endothelial cells and endothelial progenitor cells; (e) determining the activation status of the endothelial cells within the test blood specimen; and (f) correlating the amount of circulating cells and their activation status with the amount of vascular agent within the blood specimen.

Claim 23

21 . The method of claim 20 , wherein said method further comprises: (g) maintaining or modulating the dosage of subsequent administrations of the therapeutic vascular agent to the patient based on the correlation.

Claim 24

22 . The method of claim 20 , wherein the activation status of the circulating endothelial cells is determined by measuring at least one of the circulating endothelial cell activation markers selected from the group consisting of surface adhesion molecules, procoagulant factors, endothelins, growth factor receptors, nitric oxide, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), prostaglandins 12 (PGI2), tissue factors, heme oxygenase (HO), tissue plasminogen activator (tPA), mitochondria superoxide dismutase (MnSOD), Cu/Zn superoxide dismutase (Cu/Zn SOD), tumor growth factor-beta (TGF-β), cyclooxygenase 1 (COX-1), cyclooxygenase-2 (COX-2), vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), vascular endothelial growth factor (VEGF), VEGF receptor, E-selectin, and P-selectin.

Claim 25

23 . The method of claim 20 , wherein the activation status of the endothelial cells is determined by measuring at least one of the endothelial cell activation markers selected from the group consisting of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule (VCAM), and heme oxygenase (HO).

Claim 26

24 . The method of claim 20 , wherein the endothelial cell activation marker is a vasoactive compound selected from the group consisting of endothelins, nitric oxide, and prostaglandins.

Claim 27

25 . The method of claim 20 , wherein the endothelial cell activation marker is VCAM-1.

Claim 28

26 . The method of claim 20 , wherein the endothelial cell activation marker is ICAM-1.

Claim 29

27 . The method of claim 20 , wherein the endothelial cell activation marker is E-selectin.

Claim 30

28 . The method of claim 20 , wherein the therapeutic vascular agent is selected from the group consisting of angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, diuretics, alpha-adrenoreceptor antagonists, cardiac glycosides, phosphodiesterase inhibitors, beta-adrenoreceptor antagonists, calcium channel blockers, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, imidazoline receptor blockers, endothelin receptor blockers, and organic nitrites.

Claim 31

29 . The method of claim 20 , wherein the therapeutic vascular agent is an angiotensin converting enzyme inhibitor.

Claim 32

30 . The method of claim 20 , wherein the angiotensin converting enzyme inhibitor is selected from the group consisting of captopril, zofenopril, enalapril, lisinopril, quinapril, ramapril, perindopril, cilazapril, benazapril, fosinopril, trandolopril, benazeprilat, delapril, fentiapril, imidapril, libenzapril, moexipril, pentopril, perindopril, pivopril, quinaprilat, spirapril, spiraprilat, ceronapril, enalapril, indolapril, and alacepril.

Claim 33

31 . The method of claim 20 , wherein the therapeutic vascular agent is a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor.

Claim 34

32 . The method of claim 20 , wherein the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and cerivastatin.

Claim 35

33 . The method of claim 20 , wherein the patient has or is suspected of having a vascular disease.

Claim 36

34 . The method of claim 33 , wherein the vascular disease is selected from the group consisting of hypertension, hypertrophy, arrhythmia, congestive heart failure, myocardial ischemia, myocardial infarction, heart failure subsequent to myocardial infarction, ischemia reperfusion injury, blood coagulation, and platelet aggregation.

Claim 37

35 . The method of claim 20 , wherein the amount of therapeutic vascular agent within the test blood specimen is measured by an immunoassay.

Claim 38

36 . The method of claim 20 , wherein the amount of circulating endothelial cells within the test blood specimen is measured by flow cytometry.

Claim 39

37 . The method of claim 22 , wherein at least one of the endothelial cell activation markers is measured by an immunoassay.

Claim 40

38 . The method of claim 22 , wherein at least one of the endothelial cell activation markers measured is an enzyme, and wherein the activation status of the endothelial cells is determined by measuring the enzymatic activity exhibited by the enzyme.

Claim 41

39 . The method of claim 38 , wherein the enzyme is endothelial nitric oxide synthase.

Claim 42

40 . The method of claim 22 , wherein at least one of the circulating endothelial cell activation markers is an enzyme, and wherein the activation status of the circulating endothelial cells is determined by measuring the amount of the enzyme, measuring the amount of RNA encoding the enzyme, and measuring the amount of the enzymatic activity exhibited by the enzyme.

Claim 43

41 . A method of diagnosing or monitoring a vascular disease in a patient, said method comprising: (a) administering a therapeutic vascular agent to the patient; (b) obtaining a test blood specimen from the patient; (c) measuring the amount of vascular agent within the blood specimen; (d) measuring the amount of RNA encoding an endothelial cell activation marker within the test blood specimen; (e) measuring the amount of endothelial cell activation marker enzymatic activity within the test blood specimen, if the endothelial cell activation marker is an enzyme; (f) measuring the amount of endothelial cells within the test blood specimen, wherein the endothelial cells are selected from the group consisting of circulating endothelial cells, endothelial progenitor cells, and both circulating endothelial cells and endothelial progenitor cells; and (g) correlating at least one of the following with the amount of endothelial cells and the amount of therapeutic vascular agent within the test blood specimen: (i) amount of RNA encoding an endothelial cell activation marker; and (ii) amount of endothelial cell activation marker enzymatic activity.

Claim 44

41 . A method of diagnosing or monitoring a vascular disease in a patient, said method comprising: (a) administering a therapeutic vascular agent to the patient; (b) obtaining a test blood specimen from the patient; (c) measuring the amount of vascular agent within the blood specimen; (d) measuring the amount of RNA encoding an endothelial cell activation marker within the test blood specimen; (e) measuring the amount of endothelial cell activation marker enzymatic activity within the test blood specimen, if the endothelial cell activation marker is an enzyme; (f) measuring the amount of endothelial cells within the test blood specimen, wherein the endothelial cells are selected from the group consisting of circulating endothelial cells, endothelial progenitor cells, and both circulating endothelial cells and endothelial progenitor cells; and (g) correlating at least one of the following with the amount of endothelial cells and the amount of therapeutic vascular agent within the test blood specimen: (i) amount of RNA encoding an endothelial cell activation marker; and (ii) amount of endothelial cell activation marker enzymatic activity.

Claim 45

42 . The method according to claim 41 , further comprising maintaining or modulating the dosage of subsequent administrations of the therapeutic vascular agent to the patient based upon the correlation.

Claim 46

43 . The method according to claim 42 , further comprising comparing the amounts of endothelial cells and therapeutic vascular agent and at least one of the following within the test blood specimen with that of a control blood specimen: (i) amount of RNA encoding a endothelial cell activation marker; and (ii) amount of endothelial cell activation marker enzymatic activity.

Claim 47

44 . The method according to claim 41 , wherein a higher amount of RNA encoding an endothelial cell activation marker per endothelial cell or higher amount of endothelial cell activation marker enzymatic activity per unit of endothelial cells indicates the presence of vascular disease.

Claim 48

45 . A kit for monitoring vascular endothelial function comprising at least two of the components selected from the group consisting of: (a) means for measuring the amount of endothelial cells within a test blood specimen, wherein said endothelial cells are selected from the group consisting of circulating endothelial cells, endothelial progenitor cells, and both circulating endothelial cells and endothelial progenitor cells. (b) means for measuring the amount of a therapeutic vascular agent within the test blood specimen; (c) means for measuring the amount of RNA encoding an endothelial cell activation marker within the test blood specimen; and (d) means for measuring the amount of endothelial cell activation marker enzymatic activity within the test blood specimen.

Claim 49

45 . A kit for monitoring vascular endothelial function comprising at least two of the components selected from the group consisting of: (a) means for measuring the amount of endothelial cells within a test blood specimen, wherein said endothelial cells are selected from the group consisting of circulating endothelial cells, endothelial progenitor cells, and both circulating endothelial cells and endothelial progenitor cells. (b) means for measuring the amount of a therapeutic vascular agent within the test blood specimen; (c) means for measuring the amount of RNA encoding an endothelial cell activation marker within the test blood specimen; and (d) means for measuring the amount of endothelial cell activation marker enzymatic activity within the test blood specimen.

Claim 50

46 . The kit of claim 45 , wherein said means for measuring the amount of endothelial cells comprises at least one antibody or antibody fragment specific for an antigen selected from the group consisting of P1H12, CD146, CD34, VEGF receptor-2, and CD133.

Claim 51

47 . The kit of claim 45 , wherein said means for measuring the amount of RNA comprises a primer.

Claim 52

48 . The kit of claim 45 , wherein said means for measuring the amount of said therapeutic vascular agent comprises an antibody or antibody fragment that recognizes said therapeutic vascular agent.

Claim 53

49 . The kit of claim 45 , wherein said means for measuring the amount of endothelial cell activation marker enzymatic activity comprises an enzymatic substrate of the endothelial cell activation marker.