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(American Journal of Pathology. 2005;166:923-933.)
© 2005 American Society for Investigative Pathology

Matricellular Homologs in the Foreign Body Response

Hevin Suppresses Inflammation, but Hevin and SPARC Together Diminish Angiogenesis

Thomas H. Barker^*, Paul Framson^*, Pauli A. Puolakkainen, May Reed, Sarah E. Funk^* and E. Helene Sage^*

From the Hope Heart Program,^* Benaroya Research Institute at Virginia Mason, Seattle, Washington; the Department of Medicine, University of Washington, Seattle, Washington; and the Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland

	Abstract

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Implanted foreign materials, used to restore or assist tissue function, elicit an initial acute inflammatory response followed by chronic fibrosis that leads to the entrapment of the biomaterial in a thick, poorly vascularized collagenous capsule. Matricellular proteins, secreted macromolecules that interact with extracellular matrix proteins but do not in themselves serve structural roles, have been identified as important mediators of the foreign body response that includes inflammation, angiogenesis, and collagen synthesis and assembly. In this report we delineate functions of hevin and SPARC, two homologs of the SPARC family of matricellular proteins, in the foreign body response. Despite their sequence similarity, hevin and SPARC mediate different aspects of this fibrotic response. Using mice with targeted gene deletions, we show that hevin is central to the progression of biomaterial-induced inflammation whereas SPARC regulates the formation of the collagenous capsule. Although vascular density within the capsule is unaltered in the absence of either protein, SPARC-hevin double-null capsules show substantially increased numbers of vessels, indicating compensatory functions for these two proteins in the inhibition of angiogenesis. These results provide important information for further development of implant technology.

Hevin (also known as SC-1, MAST 9, SPARC-like 1, and ECM 2), originally cloned (as SC-1) from rat brain⁴ and from a human high endothelial venule endothelial cell library,^5,6 is a member of the SPARC family of matricellular proteins recently grouped on the basis of a novel, extracellular Ca⁺²-binding (E-C) module immediately preceded by a follistatin-like module.⁷ Whereas the E-C and follistatin-like domains are thought to confer activities common to the group, the family displays a poorly conserved N-terminal acidic domain, as well as unique modules specific to other members of the family.^8-14 Murine (m) hevin exhibits an amino acid identity to mSPARC of 53%; the N-terminal region accounts for its increased molecular weight, relative to SPARC (72 kd versus 32 kd), and perhaps the differences in tissue location.^15-17 In addition to inhibition of adhesion and focal adhesion formation,^5,6 hevin has also been shown to enhance B-cell lymphopoiesis¹⁸ and to interact with interstitial collagen I fibrils,¹⁴ both of which underscore the participation of hevin in nonneural cell-ECM interactions, ie, in bone marrow and connective tissue. Consistent with the phenotypes of most matricellular gene-targeted mice, hevin-null mice are viable and were initially claimed to be phenotypically normal.¹⁹

Implants (drug delivery devices, catheters, electrodes, and prostheses) derived from synthetic biomaterials have been used to restore the functionality of tissues and to act either as tissue replacements or in partial support of compromised tissue elements.²⁰ Failure of implant technology occurs most commonly throughout extended periods of time. Importantly, long-term implant solutions are lacking. Implants lose functionality as a result of a normal physiological response to foreign materials, termed the foreign body reaction (or response) (FBR).²¹ After an initial acute inflammatory response, including neutrophil and monocyte-derived macrophage recruitment, tissue surrounding implanted biomaterials exhibits a chronic fibroproliferative/fibrotic response that ultimately leads to the entrapment of the foreign material in a thick collagenous capsule.^21,22 Collagen encapsulation significantly alters the release kinetics of drug delivery devices and has been shown to be a fundamental cause of micromotion in joint replacements that leads to localized cellular apoptosis and bone resorption. Matricellular proteins have been identified as important mediators of various aspects of the FBR, ranging from the initial inflammation to the synthesis of the collagenous capsule.^23-25

In this study we have asked whether hevin, a matricellular protein related to SPARC, is a mediator of the FBR, and specifically of ECM production, the principal target of SPARC in this injury model. We report that targeted disruption of the hevin gene in mice is associated with an increased inflammatory cell response at the implant surface, with no significant effect on the collagenous capsule, whereas SPARC-null mice exhibit significantly decreased collagen encapsulation, as reported previously,²⁴ with no discernable effect on the inflammatory component. In contrast, targeted deletion of both hevin and SPARC genes in mice results in a FBR characterized by an increased inflammatory response, decreased collagen capsule thickness, and increased angiogenesis of the collagen capsule. Therefore, each homolog exhibited a unique dominant function, and possible compensatory activities with respect to the inhibition of angiogenesis. We propose that the FBR manifested in the hevin-SPARC double-null mouse is highly significant for further development of implant technology.

	Materials and Methods

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Materials and Experimental Model

Hevin/SPARC double-null mice (HvS–/–) (mixed background) were obtained from Dr. P.J. McKinnon¹⁹ (St. Jude Children’s Research Hospital, Memphis, TN) and were back-crossed five generations onto a pure 129SVE background. Wild-type (WT), hevin-null (Hv–/–), and SPARC-null (S–/–) mice were also generated by backcrossing onto a 129SVE strain (minimum of five generations). Genotypes were monitored by polymerase chain reaction on purified tail DNA. Cellulose implants were produced from Millipore filters (type HA, mixed cellulose ester, pore size 0.45 µm, 4.7 mm diameter, and 0.15 mm width; Millipore Corp., Bedford, MA) with the use of a sterile 5-mm diameter punch biopsy tool and were stored in ethanol.²⁴ Implants were incubated in sterile phosphate-buffered saline (PBS), pH 7.4, to remove the ethanol before implantation. Two cellulose disks were implanted subcutaneously on the dorsum of 3- to 7-month-old WT (n = 10), HvS–/– (n = 10), Hv–/– (n = 10), and S–/– (n = 6) mice under anesthesia. This age range represents that of normal young adult mice, and previous studies have not shown variability of results within this group.^24,25 After implantation, incisions were closed (Autoclip; Clay Adams, Parsippany, NJ), and mice were maintained for 28 days before explantation. No animals developed infection, and all implants were used in the subsequent analysis. Implants were removed en bloc and processed. All animal studies were performed according the guidelines of the American Association for Accreditation of Laboratory Care and The Fred Hutchinson Cancer Center Animal Care and Use Committee.

Immunohistochemistry

Explants were fixed in Methacarn reagent for 3 hours at room temperature, embedded in paraffin, sectioned (5 µm, 10 sequential sections), and mounted on slides. The slides were heated to 60°C for 1 hour, deparaffinized, and rehydrated. Standard chemical stains [hematoxylin and eosin, Masson’s trichrome (MT), and picrosirius red (PR)] were performed at the University of Washington Histology Core Facility. In samples processed for immunohistochemistry, endogenous peroxidase activity was quenched by a 30-minute incubation in 3% H₂O₂/PBS solution, washed, and blocked with 20% Aquablock (East Coast Biologics, Inc., North Berwick, ME) in PBS/0.2% Tween-20 for 30 minutes. Immunolocalization of hevin was performed with a rat anti-mouse hevin monoclonal antibody (mAb) (12-51²⁶ ) at 10 µg/ml. Macrophages were immunolocalized with F4/80 and CD45 antibodies (10 µg/ml) (F4/80, Serotec, Raleigh, NC; CD45, BD Biosciences Pharmingen, San Diego, CA), and blood vessels, with MECA32 hybridoma supernate (Developmental Studies Hybridoma Bank, Iowa City, IA). Detection of the primary antibodies was performed with appropriate horseradish peroxidase-conjugated secondary antibodies (1:500) followed by incubation with 3,3'-diaminobenzidine (DAB substrate kit; Vector Laboratories, Burlingame, CA). Adjacent sections were used when possible, and appropriate IgGs were used to control for background and nonspecific staining. All sections were counterstained with Hematoxylin QS (Vector Laboratories).

Histological Analysis and Quantification of the FBR

Images of the implant and surrounding tissue were acquired with a Leica DMR inverted microscope fitted with a SPOT digital camera system. Foreign body (FB) capsule thickness was quantified on MT-stained sections with ImageJ software. The average thickness of the collagen capsule (excluding inflammatory cells and sectioning artifacts) was measured by integration of the collagen-stained area throughout the entire length of the implant section. At least 30 sections from each experimental group were analyzed (with measures on both sides of the implant). Collagen capsules were further analyzed for relative collagen fiber maturity by staining with PR.

The total inflammatory cell response was quantified by determining the percentage of the implant surface lined by inflammatory cells, including FB giant cells (FBGCs), according to the formula X = L_I/L_IC. L_I is the length of the implant surface and L_IC is the length of implant surface lined by inflammatory cells, identified in MT-stained sections as cells (mono- and multinucleated) immediately adjacent to the implant surface. Immunostaining of sections (immediately adjacent to MT-stained sections) with F4/80 and CD45 was used to verify the macrophage lineage of cells identified in the quantification as giant cells and inflammatory cells. Figure panels comparing immunostaining and MT-staining represent equivalent locations (identical X, Y coordinates with respect to a constrained origin) in the adjacent sections.

Vascular indices (number of vessels/µm² of capsule) were determined by counting the number of erythrocyte-containing blood vessels and capillaries within the FB capsule in MT-stained sections.²⁴ Analysis of capsular vessel size distribution was also performed. Immunostaining with MECA32 was used to verify vascular structures. All microscopic analyses was performed in a blinded manner to eliminate investigator bias.

Statistics

A Student’s t-test was used to determine statistical significance between sample sets. P values <0.05 were considered to be statistically significant, with a confidence level of = 0.95.

	Results

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The Matricellular Protein Hevin Is Not Expressed in the FB Capsule and Does Not Contribute to Collagen Encapsulation

We have previously shown that SPARC is expressed in FB capsules and that mice lacking the SPARC gene display decreased capsule thickness and collagen content.²³ These data, and the binding of SPARC to collagen,²⁷ indicate a role for SPARC in the regulation of collagen matrix assembly. To address whether the SPARC homolog hevin has a similar distribution and/or function, we stained for hevin in FB capsules of WT mice and quantified the thickness of collagen capsules formed in Hv–/– mice compared to WT controls. Implant capsules stained with a rat anti-hevin mAb indicate that hevin is not present in FB capsules, despite its location in adjacent dermal tissue within the same section (Figure 1) . Additionally, MT-stained implant sections demonstrate that, unlike the S–/– counterparts, Hv–/– mice mount a robust encapsulation of this biomaterial (Figure 2) . In contrast, the lack of SPARC in HvS–/– (double-null) mice results in a similarly reduced capsule, compared to that of S–/– animals. Quantification of MT-stained implants (WT, n = 20; Hv–/–, n = 20; HvS–/–, n = 20; S–/–, n = 12) illustrates that WT and Hv–/– animals mount a similar response with respect to FB capsule thickness, whereas HvS–/– and S–/– mice exhibit a reduced response with respect to this parameter (Figure 3) .

View larger version (170K): [in this window] [in a new window]   Figure 1. Hevin is not present within the FB capsule. Implants from WT mice were preserved in Methacarn fixative and were sectioned. A: Isotype controls show little nonspecific staining. Murine hevin was detected with mAb 12-51 in skin adjacent to the implant section (B) but not in the FB capsule itself (C and D, implant capsules of two different WT mice). A capsular blood vessel (black arrow), inflammatory cells at the implant edge (red arrows), implant capsule (C), epidermis (E), dermis (D), and muscle (M) are indicated. Scale bar, 50 µm.

View larger version (143K): [in this window] [in a new window]   Figure 2. Hevin does not affect FB capsular thickness. Explanted cellulose wafers were fixed, sectioned, and stained with MT reagent. Mice lacking hevin (B) do not differ with respect to the collagen encapsulation response in comparison to WT controls (A). However, in the absence of SPARC (C, S–/–; D, HvS–/–), the thickness of the capsule (indicated as C) is decreased. Scale bar, 50 µm.

View larger version (29K): [in this window] [in a new window]   Figure 3. S–/– and HvS–/– mice exhibit significantly reduced capsular thickness. Collagen capsules surrounding the implant were quantified on MT-stained sections. A minimum of five images per implant (WT, Hv–/–, and HvS–/–: n = 20; S–/–: n = 12) were analyzed with ImageJ software. Data are presented as the average thickness of the collagenous capsule ± SE (***, P < 0.001; #, P < 0.05).

View larger version (59K): [in this window] [in a new window]   Figure 4. Hevin does not affect the maturation of collagen fibers in FB capsules. In PR-stained sections under polarized light, mature collagen appears red, whereas immature collagen is yellow or green. Hv–/– mice (B) are similar with respect to the maturity of the capsular collagen fibers, in comparison to WT (A). Mice lacking SPARC (C, S–/–; D, HvS–/–) display more immature collagen (white arrows) surrounding the implant, as well as diminished collagen overall. Scale bar, 10 µm.

Although primarily undefined in the context of implanted biomaterials, inflammation is the first phase of the host FBR, and these neutrophils, peripheral blood leukocytes, and macrophages influence the resolution of the FBR through the secretion of inflammatory cytokines and growth factors. Commonly, FBGCs (fused, multinucleated, macrophage-derived cells^29-31 ) are described as the predominant inflammatory cell; however, mononucleated inflammatory cells (neutrophils and macrophages) also exist at the implant surface. Because of the exuberant inflammatory cell responses that we observed, we were unable in many instances to distinguish FBGCs from mononucleated inflammatory cells at the implant edge in most of the Hv–/– and HvS–/– sections. For this reason we quantified the inflammatory response at the implant surface by determining the percentage of the implant length covered by inflammatory cells (identified by their predominant eosin staining and close apposition to the implant). Quantification indicates that mice lacking hevin mount an enhanced inflammatory cell response, whereas S–/– mice do not (Figure 5A) . In the limited number of instances in which we could identify FBGCs (by the criterion of multiple nuclei), the numbers did not appear to differ significantly among sample groups. The number of mononucleated inflammatory cells, however, was significantly increased in mice lacking hevin. Additionally, HvS–/– (Figure 5C) and Hv–/– mice (Figure 5D) displayed a higher percentage of FBGCs with protrusions or cell processes (Figure 5C) extending into the implant, in comparison to WT and S–/– mice (Figure 5B) . The mononucleated inflammatory cells present at the implant edge in Hv–/– and HvS–/– mice also showed cell processes in the cellulose material, whereas the few WT and S–/– counterpart cells did not display this characteristic. The mono- and multinuclear inflammatory cells did not strictly invade the material, ie, no entire cell bodies or nuclei were seen within the implant space. The invasive nature of these cells is evident from the cellular processes extending into the implant. Immunolocalization with CD45 (a marker of nonerythrocytic cells of hematopoietic origin) and F4/80 (a general marker of murine macrophages, but not lymphocytes and polymorphonuclear cells) in Hv–/– and HvS–/– implant sections indicates that, in addition to the FBGCs (Figure 6, E and F) , the mononucleated inflammatory cells at the implant edge are derived from a macrophage lineage. CD45 staining appears predominantly in the cell cytoplasm (Figure 6C) , whereas F4/80 was localized mainly to the extended cellular processes (Figure 6D) . Together these data indicate that hevin, despite its apparent absence from FB capsules, regulates the recruitment, activation, and/or maintenance of cells participating in the inflammation stage after biomaterial implantation. This is a novel, hevin-specific function, because S–/– animals did not exhibit this response.

View larger version (69K): [in this window] [in a new window]   Figure 5. Hevin influences the inflammatory response to implanted biomaterials. A: Hv–/– and HvS–/– mice, but not S–/– mice, demonstrate a significant increase in the percentage of implant surface covered by inflammatory cells, in comparison to WT controls (***, P > 0.0001). HvS–/– mice displayed FBGCs with cellular processes in the implant (C, black arrows), whereas this characteristic was not apparent in WT FBGC (B). Hv–/– (D) and HvS–/– (E) also displayed more mononucleated inflammatory cells with cellular processes (black arrows) in the implant, compared to WT (B). Scale bar, 10 µm.

View larger version (112K): [in this window] [in a new window]   Figure 6. Cellular populations lining the implant express CD45 and F4/80. Overlapping regions of adjacent sections (Hv–/– mice) (A–D) show mononucleated inflammatory cells at the implant edge with invasive processes (black arrows) penetrating the cellulose implant (A). These cells express CD45, a hematopoietic marker (C), and F4/80, a macrophage receptor (D). B: Control IgG is shown. In adjacent sections, FBGCs are stained for CD45 (E) and F4/80 (F). Scale bar, 10 µm.

Together, SPARC and Hevin Regulate Angiogenesis within the Collagenous Capsule

Vascularity of the collagen capsule surrounding biomaterials is significant for the long-term function of implanted devices. In our study, quantification of vascular indices (number of vessels per capsule area in µm²) illustrates that only HvS–/– mice exhibit increased vascularity of FB capsules relative to WT (Figure 7A) , despite the increased inflammation in Hv–/– mice and the reduced capsular thickness seen in S–/– genotypes. As reported previously, S–/– mice exhibit nearly equivalent vascularity compared to that of WT, despite their significantly reduced capsular thickness.²⁴ Despite the difference in blood vessel density, HvS–/– capsules had a nearly equivalent distribution of vessel diameters, compared to those of WT capsules (Figure 7B) .

View larger version (20K): [in this window] [in a new window]   Figure 7. Together, hevin and SPARC influence angiogenesis into the FB capsule. HvS–/– mice, but not WT, Hv–/–, or S–/– mice, exhibit significantly increased vascular density in FB capsules. Erythrocyte-containing vessels were counted and the vessel diameters were measured. The data are presented as the average number of blood vessels per unit capsule thickness (n/µm) ± SE (A: *, P > 0.01). The distribution of blood vessel lumen diameters does not differ between WT (solid line) and HvS–/– mice (dashed line) (B).

	Discussion

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In this study we show that Hv–/– mice manifest a response similar to that of WT mice in regard to capsular thickness as part of the FBR to implanted methylcellulose disks. In contrast, S–/– mice and HvS–/– mice exhibit significantly reduced collagenous capsules compared to those of WT and Hv–/– mice. SPARC has been shown to influence collagen deposition and fiber assembly by multiple mechanisms that include its influence on TGF-ß signaling³² and possibly steric effects through its binding to collagen I.²⁷ A significant finding is that hevin does not appear to rescue these functions in S–/– mice.²⁸

Both the Hv–/– and HvS–/– mice exhibited a substantial increase in the amount of inflammatory cells lining the implant surface and presumably extending processes into the cellulose material. Included among these were FBGCs with cellular protrusions; these protrusions could be acidified closed compartments,^33,34 although insufficient data preclude precise identification of these structures. This function appears unique to hevin and could have a major impact on the resolution of the FBR as well as wound healing and tumor progression. Notably, we found no statistically significant differences in the proliferation (PCNA- or Ki67-positive cells) or apoptotic indices (TUNEL-positive cells) in the four different genotypes with respect to the macrophage-like/FBGC populations lining the implants. The effect of hevin deletion on inflammatory cells, observed in this study, therefore appears to result from enhanced recruitment rather than increased proliferation or decreased apoptosis.

How hevin could influence the inflammatory response is unknown.³⁵ Published observations suggest possibilities: 1) production of hevin by high endothelial venules of human tonsil indicates its general expression in lymph nodes. Furthermore, the location of hevin on high endothelial venules and the de-adhesive effects of hevin on endothelial cells in culture^6,26 raise the hypothesis that hevin mediates changes in high endothelial venule architecture that facilitates the passage of lymphocytes from the blood into node compartments, where antigen presentation occurs. Thus hevin could be exerting an influence on adaptive immunity, and consequently, inflammation. 2) The prominence of hevin as a marker of tumor stromal endothelium,³⁶ and similarities between inflammation and the stromal response to solid tumors, indicate that endothelium associated with pathology generally expresses hevin.⁷ If so, hevin could regulate the congress of immune cells between the blood and areas of inflammation. However, the apparent absence of hevin in capillaries of the FBR capsule (Figure 1) requires some reconciliation with this hypothesis. 3) The augmentation of B-cell lymphopoiesis in culture by hevin and the expression of hevin by bone marrow stromal cells raise the possibility of global influences of hevin on hematopoiesis. Notably, hevin failed to augment myeloid lymphopoiesis in culture.¹⁸ Presuming the F4/80+ cells in our FBR model to be derived from a myeloid lineage, some reconciliation of our results with a hematopoietic hypothesis is required. Speculation about the role of hevin in inflammation also includes its potential influences on cytokines and chemokines at the inflammatory site, and differentiation, survival, and proliferation of inflammatory cells. Apoptotic indices and proliferation indices of the implant-lining populations at day 28 after implantation did not correlate with implant-lining inflammatory population sizes, although a definitive documentation of implant-associated apoptosis and proliferation would require a detailed temporal analysis.

Despite the enhanced chronic inflammatory response, Hv–/– mice evince only a trend toward increased collagen capsular thickness, and HvS–/– mice display a severely diminished capsular thickness (Figure 2) . These data indicate, first, that FB capsule thickness may be less affected by local inflammation than previously thought, and, second, that the effect of SPARC on collagen deposition is downstream of the influence of inflammatory cytokines or growth factors (eg, transforming growth factor-ß signaling), a contention supported by previous studies.³²

Significantly, HvS–/– mice show a statistical increase in the vascular index of the FB capsule. Unlike both Hv–/– and S–/– single-null mice, which did not differ significantly from WT controls, HvS–/– mice displayed a threefold increase in vessel density of the FB capsule. This result indicates a compensatory function of SPARC and hevin in the formation of new vessels. Both SPARC and hevin have been detected in the vascular endothelium of various tissues,^8,36 and hevin has been identified as the second most abundant pan-endothelial marker by serial analysis of gene expression of normal and tumor vascular endothelial cells.³⁶ Quantification of vessel diameters indicates that the vessel profiles of HvS–/– mice and WT controls do not differ significantly. This result is not surprising, given the relatively short time of implantation (28 days). Because it would be expected that most capsular vessels were newly formed, our data indicate that proper maturation of vessels is not impaired in the HvS–/– mice. Earlier studies using S–/– mice implied that capsular thickness might regulate the formation of new vessels (ie, less ECM in thin capsules does not support vascular ingrowth²⁴ ), but the current data refute this premise and indicate that substantial angiogenesis can occur in a thin capsule. Importantly, from a standpoint of therapy, this result raises the possibility of providing FBRs with thin yet highly vascularized collagenous capsules, which would enhance the life expectancy of implanted devices based on nondegradable biomaterials. In particular, biosensors and drug delivery devices, which must actively sense and respond to their local environment, would be more efficacious if this biology could be translated into medical technology.

Matricellular proteins, which function as modifiers of cell-ECM interactions, affect the progression and outcome of host responses to implanted biomaterials. To date every matricellular protein studied has had some effect on the FBR.³ These macromolecules are prime targets for therapeutic design because of their demonstrated impact on cell-ECM interactions during responses to injury.

	Acknowledgements

 
We thank Dr. Millicent Sullivan for advice regarding hevin staining and Sarah Funk for animal husbandry.

	Footnotes

 
Address reprint requests to E. Helene Sage, Ph.D., Hope Heart Program, Benaroya Research Institute at Virginia Mason, 1201 Ninth Ave., Seattle, WA 98101. E-mail: hsage{at}benaroyaresearch.org

Supported by the University of Washington Engineered Biomaterials National Science Foundation Engineering Research Center (grant EEC-9529161 to T.H.B., E.H.S., B.D.R.), the Helsinki University Central Hospital (research funds to P.P.), and the National Institute of General Medical Sciences (grant R01 40711 to E.H.S.).

Accepted for publication November 24, 2004.

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