Analysis of Aortic Valve Function in the Absence of Endothelial Retinoblastoma Protein
aortic valve disease (CAVD) affects 2-3% of the population age over the age of 75 years.
Surgical replacement remains the only treatment for CAVD, highlighting an acute need to
better understand CAVD biology to develop medical treatments. The pathological process
of CAVD shares phenotypic and transcriptional similarities with the physiological
process of osteogenesis. Since... read morethe retinoblastoma protein (pRb) pathway regulates the
activation state of a wide variety of mesenchymal cells and is required for proper bone
development I hypothesized that the pRb pathway regulates development and progression of
aortic valve disease. To test this hypothesis I generated a mouse model of conditional
pRb knockout (cKO) in the aortic valve by Tie2-Cre mediated excision of floxed Rb1
alleles in the endothelial lineage. Aortic valve function in Rb1fl/fl;Tie2Cre+ was
compared with control Rb1+/fl;Tie2Cre+ (pRb het) as well as mice with a loss of CDK6
kinase activity (CDK6 K43M; effectively producing a gain of pRb function) by
echocardiography. Aged pRb cKO animals had a significantly higher incidence of aortic
valve regurgitation compared to pRb het controls. By comparison, CDK6 K43M mice had
normal aortic valve function. While overt calcification was not observed, pRb cKO aortic
valves did have thicker leaflets, increased stiffness and altered collagen deposition,
all hallmarks of early CAVD. Furthermore, pRb cKO valves had increased α-SMA
compared to controls, consistent with active valve interstitial cells (VICs) that are
likely responsible for the observed extracellular matrix changes. These results reveal a
requirement for pRb in VIC quiescence and maintenance of valve function. To induce
vascular calcification, a portion of the mice were aged on a high fat diet (HFD); while
HFD did not promote aortic valve dysfunction, the pRb cKO mice were found to have
reduced weight gain with decreased diet-induced liver steatosis and lower serum
cholesterol levels. These unexpected and novel findings suggest that loss of pRb in the
endothelium protects against obesity, though a mechanism must be further investigated.
The Tie2 promoter is also active in hematopoietic stem cells (HSCs). pRb was previously
revealed as a critical regulator of HSC quiescence and its loss drives differentiation
into the myeloid lineage. Here, I demonstrate that pRb cKO mice have expanded
hematopoiesis. Additionally, I found that a number of circulating pro-inflammatory
cytokines are elevated in pRb cKO mice, possibly from the increased proportion of
monocytes seen in the bone marrow and spleen of these mice. Taken together, these
findings are consistent with a model in which enhanced systemic inflammation causes
excessive VIC activation in pRb cKO mice leading to aortic valve matrix disorganization,
aortic valve regurgitation and early changes consistent with CAVD. In this thesis, I
demonstrate a novel mouse model of age-dependent aortic valve disease and implicate pRb
in the differentiation of VICs and maintenance of extracellular matrix. I further
provide evidence that absence of endothelial pRb prevents diet-induced obesity. Further
study of this model will provide greater insight into the role that the pRb pathway
plays in endothelium and diseases relevant to the Tie2 lineage of
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Genetics.
Advisors: Gordon Huggins, and Phil Hinds.
Committee: Pamela Yelick, Alan Kopin, Grace Gill, and Giovanni Ferrari.
Keywords: Genetics, Cellular biology, and Molecular biology.read less