Transforming growth factor-beta1 mechanisms in aortic valve calcification: increased alkaline phosphatase and related events.
BACKGROUND
Aortic valve stenosis is the most frequent indication for valve replacement surgery, and is commonly associated with pathologic calcification. Previous investigations by our group have shown a strong association of transforming growth factor-beta1 (TGF-beta1)-related mechanisms with calcific aortic stenosis in both cell culture and clinical pathology studies.


METHODS
In the present investigations we sought to investigate the sequence of events involved in TGF-beta1-initiated aortic valve interstitial cell calcification in cell culture, and to study related gene expression pattern differences comparing calcific aortic stenosis surgical specimens with normal aortic valve leaflets.


RESULTS
Sheep aortic valve interstitial cells (SAVIC) in culture progressively calcified over 14 days after the addition of TGF-beta1 to a significantly greater extent than non-TGF-beta1 controls. The TGF-beta1-induced SAVIC calcification was associated with maximal levels of alkaline phosphatase by 72 hours. Annexin V positive apoptosis was increased in TGF-beta1-treated SAVIC cultures at 14 days compared with controls. Matrix metalloproteinase 9 per gel zymography was detectable only in SAVIC cultures treated with TGF-beta1 from seven days on. Matrix metalloproteinase 2 was present in all SAVIC cultures per gel zymograms, either with or without TGF-beta1, but the active form of matrix metalloproteinase 2 significantly increased over 14 days in response to TGF-beta1. Quantitative gene expression studies (re: RNA levels) of human aortic valve cusps obtained at cardiac surgery demonstrated a number of related trends, including upregulation of the expression of TGF-beta1, alkaline phosphatase, and matrix metalloproteinase 9 in calcified human aortic valves.


CONCLUSIONS
Transforming growth factor-beta1 causes SAVIC to calcify due to an early maximal increase in alkaline phosphatase activity with associated apoptotic events and increased matrix metalloproteinase 9. These TGF-beta1-related mechanistic events may be of clinical relevance based upon the gene expression pattern changes observed in calcific aortic stenosis valve cusps.
Clark-Greuel
Jocelyn N
JN
Division of Cardiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318, USA.
Connolly
Jeanne M
JM
Sorichillo
Elizabeth
E
Narula
Navneet R
NR
Rapoport
H Scott
HS
Mohler
Emile R
ER
Gorman
Joseph H
JH
Gorman
Robert C
RC
Levy
Robert J
RJ
D000328:Adult; D000368:Aged; D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D001021:Aortic Valve / Q000187:drug effects / Q000378:metabolism; D001024:Aortic Valve Stenosis / Q000139:chemically induced* / Q000201:enzymology* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002114:Calcinosis / Q000139:chemically induced* / Q000201:enzymology* / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D005260:Female; D015870:Gene Expression; D006801:Humans; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008875:Middle Aged; D012313:RNA / Q000378:metabolism; D012756:Sheep; D053773:Transforming Growth Factor beta1 / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology
D015415:Biomarkers; D053773:Transforming Growth Factor beta1; D012313:RNA; D000469:Alkaline Phosphatase; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9