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Acute-phase protein haptoglobin is a cell migration factor involved in arterial restructuring¹

DOMINIQUE P. V. DE KLEIJN^*,2, MIRJAM B. SMEETS^*,, PATRICK P. C. W. KEMMEREN^*, SAI K. LIM, BEN J. VAN MIDDELAAR^*, EVELYN VELEMA^*, ARJAN SCHONEVELD^*,, GERARD PASTERKAMP^*, and CORNELIUS BORST^*

^* Experimental Cardiology Laboratory, University Medical Center, Utrecht, The Netherlands;
Interuniversity Cardiology Institute of the Netherlands (ICIN), Utrecht, The Netherlands; and
Cardiovascular Research Institute, NUMI, The National University of Singapore, Singapore

²Correspondence: Experimental Cardiology Laboratory, University Medical Center (G02 523), Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: D.deKleijn{at}hli.azu.nl

SPECIFIC AIMS

Collagen turnover and cell migration are regarded as essential processes of arterial restructuring but components and regulatory mechanisms of arterial restructuring are still unclear. This study used subtraction PCR to identify differentially expressed mRNAs involved in blood flow-induced arterial restructuring. This revealed arterial expression of the acute-phase protein haptoglobin in the adventitial fibroblast. The function of haptoglobin was studied in vitro using migration assays and in vivo using haptoglobin knockout mice. The role of haptoglobin in collagen turnover, important for migration and arterial restructuring, was examined using matrix metalloproteinase 2 (MMP-2) and MMP-9 activity assays and cell culture.

PRINCIPAL FINDINGS

Arterial expression of haptoglobin
Subtraction PCR with rabbit mRNA isolated from the partially ligated femoral artery (flow decrease) and its contralateral artery (unchanged flow) revealed 30 clones encoding 20 different mRNAs. Clone R1045 with a sequence of 647 bp was identified as a known protein. The open reading frame coded for a partial protein of 197 amino acids (EMBL:AJ250102) with a high identity to haptoglobin reported previously. Expression of haptoglobin in arterial tissue has not been reported before. A sustained increase and a decrease in blood flow both induced haptoglobin mRNA (P<0.05) and protein expression (P<0.05) in the artery. In situ hybridization showed that haptoglobin mRNA was expressed in the arterial adventitia. Colocalization of vimentin staining and haptoglobin mRNA showed that haptoglobin was produced in adventitial fibroblasts and not in macrophages.

Haptoglobin and cell migration
To investigate the function of haptoglobin in the restructuring artery, we studied the role of haptoglobin in cell migration, an important feature in arterial restructuring. Mice wild-type (wt) and haptoglobin knockout embryonic fibroblasts were used in a Boyden chamber shown in Fig. 1 . The absence of haptoglobin in haptoglobin knockout embryonic fibroblasts resulted in slower migration (P=0.03) of the fibroblasts compared with wt and heterozygous embryonic fibroblasts (Fig. 1A ). Incubation of haptoglobin wt, heterozygous, and knockout embryonic fibroblasts treated with lipopolysaccharides (LPS) induced an increase in haptoglobin mRNA expression in wt and heterozygous fibroblasts (Fig. 1A ). This increased the number of migrating wt and heterozygous cells 1.5- to 2-fold (P=0.02) but not of haptoglobin knockout fibroblasts (Fig. 1B ). Moreover, medium from wt fibroblasts increased migration of knockout cells after LPS stimulation and demonstrates that haptoglobin is involved in cell migration (Fig. 1C ).

View larger version (27K): [in this window] [in a new window]   Figure 1. Haptoglobin and cell migration. A) Northern blot analysis of RNA (10 µg/lane) from 6 mice embryonic primary fibroblasts cell lines with different haptoglobin genotypes (+/+=wild-type,±=heterozygous, -/-=knockout). Top panels: Haptoglobin mRNA expression in primary fibroblasts of 6 different embryos without LPS (left lane) or with 10 ng/ml LPS (right lane). Bottom panels: 18S ribosomal bands of lanes in top panels. B) Migration assay with primary fibroblasts of the same embryos used in panel A. Approximately 2 x 104 cells were used per chamber with 10 ng/ml LPS (black bars) or without LPS (white bars) without chemoattractant. C) Cell migration of haptoglobin -/- fibroblasts. Haptoglobin -/- fibroblasts from 2 embryos were incubated during the migration assay without LPS (white bars), with 10 ng/ml LPS (black bars), with 10 ng/ml LPS and medium from haptoglobin -/- fibroblasts incubated 16 h with 10 ng/ml LPS (gray bars), or with 10 ng/ml LPS and medium from haptoglobin +/+ fibroblasts incubated 16 h with 10 ng/ml LPS. Haptoglobin +/+ fibroblasts were used as a positive control.

Unilateral common carotid ligation in wild-type and haptoglobin knockout mice
A mouse carotid ligation model was used to investigate whether arterial restructuring was affected in haptoglobin knockout mice, as shown in Fig. 2 . Carotid arteries were obtained at 5, 8, and 20 days after cessation of blood flow.

View larger version (74K): [in this window] [in a new window]   Figure 2. Arterial restructuring in mouse blood flow cessation model of wt and haptoglobin KO mice. A) Cross-sectional wall layer areas (mm2) 5, 8, and 20 days after unilateral ligation of the common carotid artery in wt female Balb/c mice (white bars) and female Balb/c mice with a haptoglobin null mutation (black bars). Intimal hyperplasia (IH), medial area (Media) and adventitial area (Adventitia) were measured and averaged using digital analysis of at least 8 sections per artery; n = 5–7 mice per group. Data represent mean ± SD. *Statistical significance (P<0.05). B) Hematoxylin-eosin staining of representative sections of the common carotid artery after unilateral ligation. The top 6 panels represent carotid arteries 5 days (2 panels on the left), 8 days (2 panels in the middle), and 20 days (2 panels on the right) after ligation. Bar is 50 µm. The two bottom panels are an enlargement of the carotid artery of a haptoglobin knockout (right) or a wt mouse (left) 8 days after ligation. Bar is 12.5 µm. C) Haptoglobin mRNA and protein expression in left (black bar) and right (gray bar) wt female Balb/c mice carotid arteries at 0, 3, 5, 8, and 20 days after right carotid ligation. Haptoglobin mRNA is presented as the amount of plasmid containing the haptoglobin PCR product to which it correlates in the dilution series of this plasmid used in the quantitative PCR. n = 9–10 arteries/time point. *Statistical significance (P<0.05) compared to time 0.

At 5 days, morphometry showed no differences in intimal, medial, and adventitial cross-sectional areas between wt and haptoglobin knockout mice (Fig. 2A ). Hematoxylin-eosin (HE) staining (Fig. 2B ), however, showed that morphological changes occurred in all three layers in both groups as described earlier. At 8 days, morphometry showed that the intimal and adventitial areas of the carotid arteries of the knockout mice were enlarged (Fig. 2A ) compared with the wt mice. Arteries from haptoglobin knockout mice still had a detached intimal area, abnormal smooth muscle cell structure in the media, and infiltration of inflammatory cells in all three arterial layers. The wt mice, in contrast, showed almost no intima hyperplasia, and medial smooth muscle cell morphology appeared normal. Throughout the arterial wall, no inflammatory cells were found. At 20 days, morphometry and HE staining (Fig. 2A, B ) showed no differences between carotid arteries of the haptoglobin knockout mice and wt mice. The artery contralateral to the ligated artery showed no neointima formation and no morphometric differences between wt and haptoglobin knockout mouse.

Haptoglobin mRNA and protein expression in wt female Balb/c mice carotid arteries after right carotid ligation (Fig. 2C ) showed an increase of haptoglobin mRNA and protein at 3 days in the right and left carotid artery. In the ligated right carotid artery, haptoglobin mRNA is still increased at 5 days after ligation. In this right artery, gelatinase activity is increased at 3 days (20 times, P=0.001).

Haptoglobin and collagen breakdown
We explored whether haptoglobin could affect collagen breakdown since this is an important feature for cell migration and arterial restructuring. In vitro, arterial SMCs, which produce very little haptoglobin, showed an increased production of the 80 and 30 kDa gelatin products when haptoglobin was added. As no changes in procollagens were found, we presumed that haptoglobin inhibits gelatinases such as MMP-2 and MMP-9. An in vitro gelatinase assay and an MMP-2 and -9 activity assay confirmed this by showing a decrease in MMP-2 (50%) and -9 (30%) activity when haptoglobin was added. MMP-2 and -9 activity was increased in medium of heterozygous embryonic fibroblasts compared with haptoglobin knockout cells.

CONCLUSION AND SIGNIFICANCE

Haptoglobin, an acute-phase protein, is produced mainly in the liver, where it is released into the plasma and is generally considered to be important for rapid hepatic clearance of hemoglobin from plasma. Ahaptoglobinemia, however, does not cause impaired hemoglobin clearance, a feature also seen in haptoglobin knockout mice. During hemolysis, the haptoglobin knockout mice suffer greater renal tissue damage and fail to repair damaged renal tissue. This observation points to a potential role for haptoglobin in tissue repair. Other studies of haptoglobin indicate it may be involved in other extracellular matrix-related processes. Malignant ovarian tumors and abdominal aortic aneurysms are associated with elevated haptoglobin plasma concentrations. In vitro studies indicate a role for haptoglobin in bone resorption and inflammation. Moreover, haptoglobin stimulates angiogenesis in vitro and in vivo.

Expression of haptoglobin in arterial tissue has not been reported before. A sustained increase and a decrease in blood flow induced haptoglobin mRNA and protein expression. Haptoglobin mRNA expression was found in the adventitia. Colocalization with vimentin showed that haptoglobin was produced in adventitial fibroblasts and not in macrophages.

Haptoglobin function
Incubation of mice fibroblasts with LPS increased haptoglobin expression and stimulated cell migration of wt but not of haptoglobin KO fibroblasts. Moreover, medium from wt fibroblasts increased migration of knockout cells after LPS stimulation and demonstrates that haptoglobin is involved in cell migration.

Using carotid ligation in haptoglobin knockout mice, cellular rearrangement and migration were delayed, resulting in delayed repair of the arterial wall and a prolonged inflammatory response in accordance with the time course of carotid haptoglobin expression in wt mouse. Haptoglobin expression was the highest in the ligated right carotid, which showed a strong inflammatory response at 5 days. Thus, probably because of its involvement in cell migration and arterial repair, haptoglobin has an anti-inflammatory function. Arterial SMCs showed an increased production of gelatin products when haptoglobin was added. As no changes in procollagens were found, we presumed that haptoglobin inhibits gelatinases such as MMP-2 and MMP-9, the major gelatinases in the SMC culture medium. Gelatinase activity assays and MMP-2 and -9 activity in medium of wt and haptoglobin knockout embryonic fibroblasts also point to a role for haptoglobulin in regulation of gelatinase activity.

These results point to a new concept (Fig. 3 ) in which haptoglobin induces accumulation of gelatin that serves as a temporary matrix for cell migration, analogous to the temporary fibrin matrix in angiogenesis.

View larger version (18K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized inhibition of the gelatinases MMP-2 and -9 by haptoglobin resulting in improved migration and arterial restructuring. Dashed lines indicate proteins and pathways involved in formation of gelatin.

Taken together, our data indicate that haptoglobin is expressed in arteries and involved in arterial restructuring by facilitating cell migration. Promotion of cell migration by haptoglobin may be involved in other vascular and nonvascular processes like angiogenesis, tissue repair, atherogenesis, and tumor cell invasion.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0019fje; to cite this article, use FASEB J. (May 21, 2002) 10.1096/fj.02-0019fje;.

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