Method for creating endothelial cell dysfunction in cell culture

• US 20020146679A1
• Filed: 12/18/2001
• Published: 10/10/2002
• Est. Priority Date: 01/29/2001
• Status: Abandoned Application

• Current Assignee: Robert Lee Panek, Sotirios K. Karathanasis, Zhiwu Lin
• Original Assignee: Robert Lee Panek, Sotirios K. Karathanasis, Zhiwu Lin
• Inventors: Lin, Zhiwu, Karathanasis, Sotirios K., Panek, Robert Lee
• Application Number:
  US10/025,591
• Publication Number:
  US 20020146679A1
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/5064 Endothelial cells
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Abstract

The proposed invention relates to a method for creating cell culture conditions which causes human endothelial cells in culture when exposed to a cytokine, preferably, TNFα for periods of longer than 5 days (chronic exposure) to become unresponsive to growth factors and other substances in serum. The cell culture assay system allows for screening for novel molecules that may correct endothelial dysfunction and will help identify genes that could be used to prevent or correct endothelial dysfunction with gene therapy.

Claims

1. A method for simulating endothelial cell dysfunction, said method comprising culturing endothelial cells together with a physiological protein matrix, growth media, and an effective amount of at least one proinflammatory cytokine sufficient to disrupt normal response of the endothelial cells to the growth media.

2. A method according to claim 1, wherein the cytokine is tumor necrosis factor α

(TNFα

).

3. A method according to claim 1, wherein the physiological protein matrix is a basement membrane protein matrix.

4. A method according to claim 3, wherein the basement membrane protein matrix is fibrin, collagen, or MATRIGEL®

.

5. A method according to claim 1, wherein the growth media includes serum.

6. A method according to claim 1, wherein the loss of normal response comprises impaired angiogenesis.

7. A method according to claim 1, wherein the endothelial cells are cultured in the presence of the cytokine for period of time sufficient to impair angiogenesis.

8. A method according to claim 7, wherein the period of time is at least 3 days.

9. A method for identifying genes involved in endothelial cell dysfunction, said method comprising the steps of:

(a) culturing endothelial cells together with a physiological protein matrix, growth media including serum, and at least one proinflammatory cytokine for a period of time sufficient to disrupt normal endothelial function; and

(b) measuring expression of at least one selected gene, wherein altered expression of the at least one selected gene, relative to endothelial cells not contacted with the proinflammatory cytokine, indicates that the selected gene is involved in endothelial cell dysfunction.

10. A method according to claim 9, wherein the endothelial cells are cultured in the presence of the cytokine for period of time sufficient to impair angiogenesis.

11. A method according to claim 9, wherein the physiological protein matrix is a basement membrane protein matrix.

12. A method according to claim 11, wherein the basement membrane protein matrix is fibrin, collagen, or MATRIGEL®

.

14. A method according to claim 13, wherein the angiogenic activity is growth of new vascular tissue.

13. A method for determining whether candidate compound is useful for modulating angiogenesis, said method comprising the steps of:

(a) culturing endothelial cells together with a physiological protein matrix, growth media including serum, at least one proinflammatory cytokine, and a candidate compound for a period of time sufficient to disrupt normal endothelial function; and

(b) measuring the angiogenic actively of the endothelial cells, wherein altered angiogenic activity, relative to endothelial cells treated as in Step (a) not contacted with the candidate compound, indicates that the candidate compound modulates angiogenesis.

15. A kit comprising, in compartmentalized form, at least one first compartment containing at least one physiological protein matrix for culturing endothelial cells thereon, at least one second compartment containing growth media including serum, and endothelial cells for addition to the at least one physiological protein matrix;

a third compartment containing at least one proinflammatory cytokine for addition to either said first compartment of said second compartment.