Qing Song

DNA polymorphisms in the 5′-flanking region of the human tissue kallikrein gene

Abstract Human tissue kallikrein gene polymorphisms were identified in the promoter region by polymerase chain reaction (PCR) and DNA sequencing. One polymorphic region was identified between nucleotides –121 and –133 with respect to the transcription initiation site of the tissue kallikrein gene. Ten alleles with length and nucleotide sequence variations were detected among 108 unrelated Caucasians, African-Americans, and Asians. The polymorphisms show Hardy-Weinberg equilibrium. Allele-specific amplification and PCR analyses were used to detect the various forms of polymorphism. The promoter activity was analyzed in human embryonic kidney 293 cells by transient transfection assays. Sequential 5′-deletion analysis of the tissue kallikrein gene promoter revealed that the region from –144 to –98 is crucial for its promotor activity, while alleles D and H had significantly lower promoter activities than the other alleles in the –940/+10 deletion constructs. The high variability and the proximity to the tissue kallikrein gene render it suitable for application as a new tool in genetic studies for evaluation of the tissue kallikrein gene in the pathogenesis of human essential hypertension.

High Level of Circulating Human Tissue Kallikrein Induces Hypotension in a Transgenic Mouse Model

We established a unique transgenic mouse model in liver-targeted expression of human tissue kallikrein using a mouse albumin enhancer and promoter. Northern blot analysis and ELISA showed that human tissue kallikrein was predominantly expressed in the liver of transgenic mice and secreted into the circulation at a high level. The transcript was also detected in the kidney, pancreas, salivary gland and heart at a low level by reverse transcription-polymerase chain reaction followed by Southern blot analysis. Systolic blood pressures were measured by the tail-cuff method, all three independent transgenic mouse lines are hypotensive (84.6 +/- 1.0 mmHg, n = 17; 84.5 +/- 1.5 mmHg, n = 9; 83.1 +/- 0.8 mmHg, n = 13, P < 0.01) compared with the control mice (100.9 +/- 0.9 mmHg, n = 17). Administration of aprotinin, a potent tissue kallikrein inhibitor or Hoe 140, a bradykinin receptor antagonist, restored the blood pressure of transgenic mice but had no significant effect on control littermates. These studies show that over-production of tissue kallikrein in the circulation plays a role in blood pressure regulation.

Kallistatin Stimulates Vascular Smooth Muscle Cell Proliferation and Migration In Vitro and Neointima Formation in Balloon-Injured Rat Artery

Kallistatin, a serine proteinase inhibitor (serpin), is expressed in the endothelial and smooth muscle cells of blood vessels. The potential function of kallistatin in vascular biology was investigated by studying its role in the proliferation and migration of cultured primary aortic vascular smooth muscle cells (VSMCs) in vitro and in neointima formation in rat artery after balloon angioplasty in vivo. Exogenous kallistatin induced a >2-fold increase of VSMC proliferation and cell growth as measured by [(3)H]thymidine incorporation and cell counts and a 2.3-fold increase of cell migration in modified Boyden chambers. In balloon-injured vessels, endogenous kallistatin mRNA and protein levels increased up to 10-fold as determined by competitive polymerase chain reaction and by ELISA. Intense staining of kallistatin mRNA was identified in the proliferating VSMCs of balloon-injured arteries during cell migration from media to neointima by in situ hybridization histochemistry and immunohistochemistry. We observed an induction of kallistatin expression by platelet-derived growth factor (PDGF) and upregulation of p42/44 mitogen-activated protein kinase (MAPK) activity by kallistatin in cultured VSMCs. Conversely, adenovirus-mediated transfer of kallistatin antisense cDNA into cultured VSMCs inhibited PDGF-induced p42/44 MAPK activity and cell proliferation. Furthermore, local delivery of adenovirus carrying kallistatin antisense cDNA significantly downregulated kallistatin mRNA levels and attenuated neointima formation in balloon-injured rat arteries in vivo. These results indicate that kallistatin may play an important role in mediating PDGF-induced MAPK pathway on VSMC proliferation and in neointima formation after balloon angioplasty.

High level expression of human tissue kallikrein in the circulation induces hypotension in transgenic mice

In order to create an animal model expressing a high level of tissue kallikrein in the circulation, the human tissue kallikrein gene was placed under the control of a mouse albumin enhancer and promoter to target its expression to liver. Three lines of transgenic mice carrying the human tissue kallikrein gene were established. The major site of human tissue kallikrein synthesis was identified in the liver of transgenic mice, and a high level of human tissue kallikrein was secreted into the mouse circulation. The systolic blood pressures of these transgenic mice are about 15-20 mmHg lower than that of the control mice. Administration of aprotinin, a potent tissue kallikrein inhibitor, restored normal blood pressure in these animals. These studies show that a high level of foreign tissue kallikrein in the circulation plays a role in blood pressure regulation.