We are now examining, using gene expression chips, whether Stat3 can regulate its own levels of expression through a feedback mechanism controlled by certain guanine exchange factors. We are also using intact mouse models to determine whether blocking Stat3 activation can reduce post-ischemic inflammation and tissue injury. We are also interested in identifying other mechanisms for ICAM-1 regulation in endothelial cells, and discovered a novel pathway involving the chemokine fractalkine interacting with specific fractalkine receptors on the endothelial cell membrane (which were previously not known to exist), and the intracellular Jak-Stat5 pathway. This work has been spearheaded by Dr. Xiao Ping Yang.
Vascular endothelial cells express the fractalkine receptor, CX3CR1. Confocal
image shows that this receptor is located on the cell membrane and to a lesser
extent in the cytoplasm. Hypoxia-reoxygenation releases soluble fractalkine
(s-FKN), with interacts with CX3CR1 on the membrane to upregulate intercellular
adhesion molecule-1 (ICAM-1) (see brown staining in mouse heart tissue).
Dr. Yang has also been interested for many years in the role of the lipoprotein scavenger receptor SR-B1 in regulating blood lipid levels. In collaboration with the GeneSTAR Research Program, a large prospective study of families with premature coronary artery disease existing in the Division of General Internal Medicine under the direction of Dr Diane Becker, she has made the novel observation that SR-B1 regulates Lp(a) cholesterol levels through uptake of cholesterol from Lp(a). She has found several novel mutations in the SR-B1 gene that is associated with markedly elevated Lp(a) levels as well as HDL levels. This novel phenotype of combined elevations in HDL and Lp(a) is associated with a variety of previously unknown mutations in the SR-B1 gene that modify the function of the receptor. She is continuing to work on the basic biology of SR-B1 receptors in cultured cells.
The laboratory has also collaborated with the GeneSTAR Research Program in performing novel biological studies of genes found to regulate platelet function or the occurrence of coronary artery disease. The GeneSTAR Program has performed genome-wide association studies (GWAS) involving 2.5 million genotyped and imputed single nucleotide polymorphisms (SNPs) in over 3000 subjects from families with premature coronary artery disease to look for genetic variants responsible for incident coronary disease phenotypes, as well as other characterized phenotypes.
For example, GeneSTAR discovered that a variant in intron1 of the newly described gene platelet endothelial aggregation receptor-1 (PEAR1) is strongly associated with platelet aggregation to multiple agonists. We determined that subjects carrying the minor allele had reduced expression of PEAR1 protein on their platelets, and that the variant is located at a leucine zipper binding site which binds to the transcription factor C/EBP. An intron1 construct carrying the minor allele appears to bind less tightly than normal, perhaps explaining the reduced protein expression. The concept of a transcriptional promoter site in an intron of a gene is very new.
Dr. Zheqin (Paul) Cai is investigating the biological pathways responsible for ischemic preconditioning, which results in profound resistance of the myocardium to subsequent ischemic damage. While a postdoctoral fellow at Hopkins in Gregg Semenza’s laboratory, he discovered that hearts exposed to erythropoietin exhibited HIF-1 dependent protection against ischemia-reperfusion injury, and that this was mediated through phosphatitdylinositol-3-kinase signaling.
These studies have provided a scientific basis for ongoing translational trials of erythropoietin therapy in patients with acute myocardial infarction. He has found more recently that myocardial preconditioning is mediated by degradation of the protein phosphatase PTEN, a negative regulator of the serine-threonine kinase Akt. He developed a state of the art in-vivo mouse model for measuring myocardial infarct size and is using it to determine whether deletion of the PTEN gene in the heart produces a preconditioned-like state with resistance to ischemic injury (see Figure below).
He uses, in addition to the in vivo model, a Langendorff perfused heart model, and various molecular biology techniques. He has also generated mice with conditional Rac1 deletions (these have impaired preconditioning) and studied mice with knockout of the proteasome LMP-2 ? subunit (cannot be preconditioned). The results are consistent with the hypothesis that the proteasome is responsible for PTEN degradation during ischemia, which in turn leads to an increase in phosphorylated Akt and increased myocardial survival. These and related studies may lead to new therapies for myocardial protection.
Hearts from muscle-specific Pten knockout mice (Ptenlp/l ; mck-Cre+/-) and wildtype mice(Ptenlp/l ; mck-Cre-/-).
The conditional Pten knockout mice were generated by the cre-loxp technique.
Cre recombinase expression is controlled by the muscle creatine kinase promoter.
A: Muscle-specific Pten knockout mice exhibit cardiac hypertrophy.
B: PTEN protein is detectable in insterstitial cells but not in myocytes in conditional knockout mouse heart.