ARTICLE |
Correspondence to: Julie Chao, Dept. of Biochemistry and Molecular Biology, Medical U. of South Carolina, 171 Ashley Ave., Charleston, SC 29425.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue kallikrein releases kinins by specific proteolysis, an activity inhibited by kallistatin. In this study, kallikrein and kallistatin were localized to endothelial and smooth muscle cells of large, medium, and small normal blood vessels by immunohistochemical techniques. Immunostaining for both proteins was strong in the endothelium of all sizes of blood vessels and was more intense in medial smooth muscle cells of small and medium-sized blood vessels than in elastic arteries. The sites of synthesis by endothelial and smooth muscle cells were demonstrated in normal blood vessels of all sizes by in situ hybridization histochemistry. Kallikrein and kallistatin levels were measured by immunoassays in homogenates of human aorta, vena cava, and iliac artery and vein. Tissue kallikrein and kallistatin transcripts were identified in human blood vessels by RT-PCR followed by Southern blot analysis with specific oligonucleotide probes. The results demonstrated the expression and co-localization of tissue kallikrein and kallistatin in human vessels and suggest a potential role of kallistatin in regulating tissue kallikrein in blood vessels. (J Histochem Cytochem 47:221228, 1999)
Key Words: tissue kallikrein, kallistatin, endothelial cell, smooth muscle cell, localization
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue kallikrein is a serine proteinase that cleaves kininogen to release the kinin peptides bradykinin and lys-bradykinin. Kinins are locally active hormones that participate in the regulation of local blood flow, decrease peripheral vascular resistance, and increase vascular permeability (reviewed in
Human kallistatin is a novel member of the serine proteinase inhibitor (serpin) superfamily which was purified, cloned, and characterized in our laboratory (reviewed in
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue Samples for Immunohistochemistry and In Situ Hybridization
Four-µm sections were cut from formalin-fixed, paraffin-embedded archival surgical specimens. Vessels were examined from a number of anatomic sites, including the pancreas, small intestine, salivary gland, kidney, adrenal glands, aorta, splenic and renal artery and vein, iliac artery, spleen, lymph node, lung, and loose connective tissue, obtained during surgery for metastatic disease, trauma, or reconstruction.
Antibodies for Immunohistochemistry
Details of the isolation and characterization of the monoclonal antibody (MAb) G4C10, specific for human kallistatin, has been published previously (1-anti-trypsin,
1-anti-chymotrypsin, and rat kallikrein binding protein. The specificity of the rabbit antiserum against human tissue kallikrein has been previously published (
-smooth muscle actin (Dako; Carpinteria, CA), respectively, were used.
Immunohistochemistry
Immunohistochemistry was performed using the TechMate 500 robotic workstation (Ventana; Tucson, AZ) for automated histochemistry. The Steam Heat Induced Epitope Retrieval and MIP (a standard immunoperoxidase procedure using avidinbiotinperoxidase complex) protocols and ChemMate reagents were used as directed by the manufacturer (Ventana). Color development was performed with diaminobenzidine tetrahydrochloride provided with the ChemMate reagents. For kallistatin immunohistochemistry, MAb G4C10 was used at dilutions of 1:4001:800. Equally diluted mouse control ascites fluid was used as negative control. Rabbit anti-human tissue kallikrein antiserum was used at dilutions of 1:1200 and 1:2000 to detect kallikrein, and equal dilutions of normal rabbit serum served as negative controls. In control experiments, preabsorption of tissue kallikrein antiserum with purified human tissue kallikrein eliminated immunoreactivity.
In Situ Hybridization Histochemistry
In situ hybridizations specific for kallistatin and tissue kallikrein were performed as previously reported (
Collection of Vessels for ELISA and RT-PCR
Blood vessels removed at autopsy 612 hr after death were obtained from the Pathology Department of the Medical University of South Carolina. Total RNA was extracted with Trizol reagent according to the manufacturer's directions (Life Technologies; Rockville, MD). Blood vessels for ELISA were rinsed, minced, and homogenized in PBS, pH 7.4, at 4C. Homogenates were initially centrifuged at 600 x g for 20 min. Supernatants were incubated for 30 min at 4C in 0.5% sodium deoxycholate and centrifuged at 10,000 x g for 30 min, and the pellets discarded. Protein concentration was determined by the Lowry method (
Tissue Kallikrein and Kallistatin Immunoassays
ELISAs were performed using specific polyclonal antibodies, as previously described, against either tissue kallikrein (
RT-PCR/Southern Blot Analysis for Human Tissue Kallikrein and Kallistatin mRNAs
Reverse transcription (RT) was performed with Moloney murine leukemia virus reverse transcriptase (Life Technologies), 2 µg of total RNA, and 100 nmol random hexamers (Life Technologies) according to the manufacturer's protocol. Polymerase chain reaction (PCR) was performed on 1 µl of the first-strand cDNA in a 50-µl reaction mixture using primers that were designed from unique sequences of human tissue kallikrein (5' primer, AACACAGCCCA-GTTTGT; 3' primer, CTTCAC-ATAAGACAGCAC), human kallistatin (5' primer, CTATGGTTTCGTGGGTC; 3' primer, GGCACAATCCAGCTTATC), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5' primer, ATCCCATCACCATCTTCCAG; 3' primer, CCTGCTTCACCA-CCTTCTTG). All sets of primers were designed to cross intronexon boundaries. For tissue kallikrein and kallistatin amplifications of 30 PCR cycles (94C, 1 min; 55C, 1 min; 72C, 1 min) were performed in a thermal cycler, whereas for GAPDH 25 cycles (94C, 1 min; 60C, 1 min; 72C, 1 min) were performed. The PCR product was subjected to Southern blot analysis with a nested oligonucleotide probe specific to the respective gene (tissue kallikrein, GACCTCAAATCCCTGCC; kallistatin, CTCTGGAGTCAGAACCTC; GAPDH, GACCACAGTCCATGCCAT). Hybridization was carried out as previously described (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cellular Localization and Expression of Tissue Kallikrein and Kallistatin in Normal Vessels
At low power, the immunohistochemical reactivity for both tissue kallikrein (Figure 1A) and kallistatin (Figure 1D) was more intense in the endothelium and the vasa vasorum than in the media. Overall, the tunica media was more intensely stained for kallistatin than for tissue kallikrein. At higher power, distinct cytoplasmic reactivity was discernible in aortic vascular smooth muscle cells and endothelial cells, and there were increased numbers of medial smooth muscle cells positive for kallistatin (Figure 1E) compared to kallikrein (Figure 1B). Immunohistochemistry was done with anti-von Willebrand factor antibody to identify endothelial cells (Figure 1C). The identity of vascular smooth muscle cells was ascertained by immunostaining for -smooth muscle actin (Figure 1F). No reactivity was observed when normal rabbit serum was used as a negative control for the rabbit antiserum specific for human tissue kallikrein (not shown) or when anti-kallikrein antiserum was preabsorbed with purified kallikrein (not shown). In addition, reactivity was absent when mouse ascites fluid was used as negative control for the MAb specific for human kallistatin (not shown).
|
Figure 2 illustrates the similar immunohistochemical reactivity for tissue kallikrein (top row) and kallistatin (middle row) that was typical in normal medium-sized (muscular) and small vessels. In comparison to the aorta (Figure 1), smooth muscle cells in the tunica media of medium-sized vessels and arterioles were more reactive for both proteins. The higher magnification for medium vessels showed nuclei of smooth muscle cells that were often outlined by the intense cytoplasmic staining for both tissue kallikrein (Figure 2A and Figure 2B) and kallistatin (Figure 2D and Figure 2E). As in the aorta, there was strong cytoplasmic reactivity for tissue kallikrein and kallistatin in endothelial cells of medium and small arteries and veins. Reactivity was absent when normal rabbit serum was used as a negative control (Figure 2GI) for the rabbit antiserum specific for human tissue kallikrein or when anti-kallikrein antibody was preabsorbed with purified kallikrein (not shown). A similar absence of reactivity was seen for control mouse ascites fluid (not shown), used as negative control for the MAb specific for human kallistatin.
|
Figure 3 shows the in situ localization of tissue kallikrein and kallistatin mRNA revealed by using specific digoxygenin-labeled anti-sense riboprobes. Reactivity indicated that both tissue kallikrein (Figure 3A and Figure 3B) and kallistatin (Figure 3C and Figure 3D) mRNAs are synthesized by endothelial and smooth muscle cells in the aorta (Figure 3A and Figure 3C) and small vessels (Figure 3B and Figure 3D). Medium vessels (not shown) displayed positive staining in endothelial and smooth muscle cells similar to the staining seen in aorta and small muscular artery (SMA) in this figure. Reactivity was absent when a sense riboprobe was used for hybridization for tissue kallikrein (Figure 3E and Figure 3F) or kallistatin (not shown). In addition, no staining was seen when sections were treated with RNase A before hybridization with the respective anti-sense probes (not shown). These results demonstrate the specificity of labeled anti-sense probes for kallistatin or kallikrein transcripts.
|
Immunoreactive Tissue Kallikrein and Kallistatin Levels in Human Blood Vessels
Table 1 shows the levels of immunoreactive human tissue kallikrein and kallistatin in aorta, vena cava, and iliac (muscular) artery and vein measured by ELISA. Human tissue kallikrein levels are greater in the muscular (iliac) vessels than in large, elastic (aorta and vena cava) vessels. In all vessels examined, kallistatin is present in excess of tissue kallikrein. Figure 4 shows a typical curve for the tissue kallikrein standard and curves for immunoreactive kallikrein in serially diluted homogenates of aorta, vena cava, and iliac artery or vein. Dose-dependent curves of sample homogenates are parallel with the standard curve, indicating their immunological identity. Figure 5 shows curves for the kallistatin standard and for immunoreactive kallistatin in the aorta, vena cava, and iliac artery and vein. Lines for the serial dilutions of these samples are also parallel to the standard displacement curve, indicating immunological identity to purified kallistatin used as standard.
|
|
|
Identification of Tissue Kallikrein and Kallistatin Transcripts in Human Vessels by RT-PCR/Southern Blot Analysis
Figure 6 shows the identification of tissue kallikrein and kallistatin mRNAs in human vessels (aorta, vena cava, iliac artery and vein) as determined by specific RT-PCR amplification and verified by Southern blot analysis. The band migrations were consistent with the expected product sizes and were the same as amplified tissue kallikrein or kallistatin cDNA controls (not shown), indicating that the PCR products were not amplified from genomic DNA. Integrity of the RNAs used in the RT-PCR reactions was evaluated by RT-PCR/Southern analysis for glyceraldehyde-3-phosphate dehydrogenase, a constitutively expressed gene. These results suggest that tissue kallikrein and kallistatin are synthesized locally in vessels and support the results of our in situ hybridization.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study is the first to demonstrate both the immunohistochemical localization and the in situ synthesis of tissue kallikrein and kallistatin in endothelial and vascular smooth muscle cells of human large, medium and small blood vessels. Our immunohistochemical localization of tissue kallikrein in human vessels is consistent with a recent localization by Raidoo and co-workers (1997), except that they did not detect kallikrein in medial smooth muscle cells of large elastic vessels. The discrepancy may be attributed to the fact that their samples were obtained from autopsy and may have undergone some autolysis, rendering medial kallikrein undetectable. Tissue kallikrein and kallistatin are expressed in cultured endothelial cells (
The observation that tissue kallikrein appears more pronounced in human medium and small vessels is consistent with previous results revealing a similar pattern for the kininogen substrate (
Kinins also contribute significantly to the inflammatory response by mediating vasodilatation and increasing vascular permeability (
Kallistatin is a heparin-binding serpin, and its interaction with tissue kallikrein is markedly attenuated by heparin binding (
![]() |
Acknowledgments |
---|
Supported by National Institutes of Health grants HL 44083 and HL 29397.
We wish to thank Dr Jo Ann Simson for expert technical advice and Dr Gary Richards for critical evaluation of the manuscript.
Received for publication May 28, 1998; accepted October 6, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bhoola KD, Figueroa CD, Worthy K (1992) Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmocol Rev 44:1-80[Medline]
Bossaller C, AuchSchwelk W, Grafe M, Graf K, Baumgarten C, Fleck E (1992) Effects of converting enzyme inhibition on endothelial bradykinin metabolism and endothelium-dependent vascular relaxation. Agents Actions Suppl 38:171-177[Medline]
Carlson TH, Kolman MR, Piepkorn M (1995) Activation of antithrombin III isoforms by heparan sulphate glycosaminoglycans and other sulphated polysaccharides. Blood Coagul Fibrinol 6:474-480[Medline]
Chao J, Chao L (1995) Biochemistry, regulation and potential function of kallistatin. Biol Chem Hoppe Seyler 376:705-713[Medline]
Chao J, Schmaier A, Chen LM, Yang Z, Chao L (1996) Kallistatin, a novel human tissue kallikrein inhibitor: levels in body fluids, blood cells, and tissues in health and disease. J Lab Clin Med 127:612-620[Medline]
Chao JL, Stallone JN, Liang YM, Chen LM, Wang DZ, Chao L (1997) Kallistatin is a potent new vasodilator. J Clin Invest 100:11-17
Chen LM, Chao L, Chao J (1997a) Adenovirus-mediated delivery of human kallistatin gene reduces blood pressure of spontaneously hypertensive rats. Hum Gene Ther 8:341-347[Medline]
Chen LM, Chao L, Chao J (1997b) Beneficial effects of kallikrein-binding protein in transgenic mice during endotoxic shock. Life Sci 60:1431-1435[Medline]
Chen LM, Song Q, Chao L, Chao J (1995) Cellular localization of tissue kallikrein and kallistatin mRNAs in human kidney. Kidney Int 48:690-697[Medline]
Cockcroft JR, Chowienczyk PJ, Brett SE, Bender N, Ritter JM (1994) Inhibition of bradykinin-induced vasodilation in human forearm vasculature by icatibant, a potent B2-receptor antagonist. Br J Clin Pharmacol 38:317-321[Medline]
Dixon BS, Dennis MJ (1997) Regulation of mitogenesis by kinins in arterial smooth muscle cells. Am J Physiol 42:C7-20
Figueroa CD, Gonzalez CB, MullerEsterl W, Bhoola KD (1992) Cellular localization of human kininogens. Agents Actions Suppl 38:617-626[Medline]
Figueroa CD, MacIver AG, Bhoola KD (1989) Identification of a tissue kallikrein in human polymorphonuclear leucocytes. Br J Haematol 72:321-328[Medline]
Groves P, Kurz S, Just H, Drexler H (1995) Role of endogenous bradykinin in human coronary vasomotor control. Circulation 92:3424-3430
Hecker M, Porsti I, Bara AT, Busse R (1994) Potentiation by ACE inhibitors of the dilator response to bradykinin in the coronary microcirculation: interaction at the receptor level. Br J Pharmacol 111:238-244[Abstract]
Hornig B, Drexler H (1997) Endothelial function and bradykinin in humans. Drugs 54:42-47[Medline]
Hornig B, Kohler C, Drexler H (1997) Role of bradykinin in mediating vascular effects of angiotensin-converting enzyme inhibitors in humans. Circulation 95:1115-1118
Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 93:265-275
Ma JX, Song Q, Hatcher HC, Crouch RK, Chao L, Chao J (1996) Expression and cellular localization of the kallikrein-kinin system in human ocular tissues. Exp Eye Res 63:19-26[Medline]
Madeddu P, Gherli T, Bacciu PP, Maioli M, Glorioso N (1993) A kallikrein-like enzyme in human vascular tissue. Am J Hypertens 6:344-348[Medline]
Madeddu P, Varoni MV, Palomba D, Emanueli C, Demontis MP, Glorioso N, DessFulgheri P, Sarzani R, Anania V (1997) Cardiovascular phenotype of a mouse strain with disruption of bradykinin B2 receptor gene. Circulation 96:3570-3578
Nolly HL, Lama MC, Carretero OA, Scicli AG (1992) The kallikrein-kinin system in blood vessels. Agents Actions Suppl 38:1-9[Medline]
Nolly HL, Scicli AG, Scicli G, Carretero OA (1985) Characterization of a kininogenase from rat vascular tissue resembling tissue kallikrein. Circ Res 56:816-821[Abstract]
Oza NB, Schwartz JH, Goud HD, Levinsky NG (1990) Rat aortic smooth muscle cells in culture express kallikrein, kininogen, and bradykininase activity. J Clin Invest 85:597-600[Medline]
Proud D, Vio CP (1993) Localization of immunoreactive tissue kallikrein in human trachea. Am J Respir Cell Mol Biol 8:16-19[Medline]
Raidoo DM, Ramsaroop R, Naidoo S, MullerEsterl W, Bhoola KD (1997) Kinin receptors in human vascular tissue: their role in atheromatous disease. Immunopharmacology 36:153-160[Medline]
Saed GM, Carretero OA, MacDonald RJ, Scicli AG (1990) Kallikrein messenger RNA in rat arteries and veins. Circ Res 67:510-516[Abstract]
Shirk RA, Church FC, Wagner WD (1996) Arterial smooth muscle cell heparan sulfate proteoglycans accelerate thrombin inhibition by heparin cofactor II. Arterioscler Thromb Vasc Biol 16:1138-1146
Wang DZ, Song Q, Chen LM, Chao L, Chao J (1996) Expression and cellular localization of tissue kallikrein-kinin system in human adrenal gland. Am J Physiol 271:F709-716
Wiemer G, Scholkens BA, Linz W (1994) Endothelial protection by converting enzyme inhibitors. Cardiovasc Res 28:166-172[Medline]
Xiong W, Tang CQ, Zhou GX, Chao L, Chao J (1992) In vivo catabolism of human kallikrein-binding protein and its complex with tissue kallikrein. J Lab Clin Med 119:514-521[Medline]
Zhou GX, Chao L, Chao J (1992) Kallistatin: a novel human tissue kallkrein inhibitor. Purification, characterization, and reactive center sequence. J Biol Chem 267:25873-25880
Ziche M, Morbidelli L, Masini E, Granger H, Geppetti P, Ledda F (1993) Nitric oxide promotes DNA synthesis and cyclic GMP formation in endothelial cells from postcapillary venules. Biochem Biophys Res Commun 192:1198-1203[Medline]