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Collagen fiber realignment is one mechanism by which tendon responds to load. Re-alignment is altered when the structure of tendon is altered, such as in the natural process of aging or with alterations of matrix proteins, such as proteoglycan expression. While changes in re-alignment and mechanical properties have been investigated recently during development, they have not been studied in (1) aged tendons, or (2) in the absence of key proteoglycans. Collagen fiber re-alignment and the corresponding mechanical properties are quantified throughout tensile mechanical testing in both the insertion site and the midsubstance of mouse supraspinatus tendons in wild type (WT), decorin-null (Dcn(-/-)), and biglycan-null (Bgn(-/-)) mice at three different ages (90 days, 300 days, and 570 days). Percent relaxation was significantly decreased with age in the WT and Dcn(-/-) tendons, but not in the Bgn(-/-) tendons. Changes with age were found in the linear modulus at the insertion site where the 300 day group was greater than the 90 day and 570 day group in the Bgn(-/-) tendons and the 90 day group was smaller than the 300 day and 570 day groups in the Dcn(-/-) tendons. However, no changes in modulus were found across age in WT tendons were found. The midsubstance fibers of the WT and Bgn(-/-) tendons were initially less aligned with increasing age. The re-alignment was significantly altered with age in the WT tendons, with older groups responding to load later in the mechanical test. This was also seen in the Dcn(-/-) midsubstance and the Bgn(-/-) insertion, but not in the other locations. Although some studies have found changes in the WT mechanical properties with age, this study did not support those findings. However, it did show fiber re-alignment changes at both locations with age, suggesting a breakdown of tendon's ability to respond to load in later ages. In the proteoglycan-null tendons however, there were changes in the mechanical properties, accompanied only by location-dependent re-alignment changes, suggesting a site-specific role for these molecules in loading. Finally, changes in the mechanical properties did not occur in concert with changes in re-alignment, suggesting that typical mechanical property measurements alone are insufficient to describe how structural alterations affect tendon's response to load.

Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera. The latter are much larger and more heterodisperse. This high content of type V collagen in cornea is reflected by an equally elevated content of alpha1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any alpha1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human alpha1(V) transcript. The results were evaluated by reverse transcript-polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human alpha1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10-14 h the heterokaryon-containing cultures showed de novo appearance or upregulation of human alpha1(V) chain mRNA, compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human transcript. Thus, corneal cells do contain positive regulators for alpha1(V) chain mRNA, and this effect is at least somewhat cell specific.

Corneal development involves the synthesis and assembly of a number of specialized extracellular matrices. These matrices have distinctive properties derived from a unique assembly of collagens, proteoglycans and glycoproteins. The synthesis of each of these requires a number of enzymes. By probing a corneal cDNA library for genes that appeared to be up-regulated in cornea we have isolated a cDNA that represents an mRNA encoding the enzyme β- 1,4-galactosyltransferase. In cornea, a major function for this enzyme is likely to be in the synthesis of the keratan sulfate proteoglycan, lumican. Employing quantitative reverse transcript-polymerase chain reaction, we have observed that the steady-state level of mRNA for the molecule is elevated during certain stages of corneal development. It is also elevated in corneal fibroblasts in culture that have a greatly decreased synthesis of the mature lumican molecule. These data are consistent with, and complement, studies by others that show a corresponding regulation of the lumican core protein during development and in corneal fibroblast cultures.

A number of factors have been implicated in the regulation of tissue-specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta-galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large-diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino-terminal domain of type V collagen was associated with the small-diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.

De novo donor-specific antibodies ( dn DSA) that develop after renal transplantation are independent predictors of allograft loss. However, it is unknown if dn DSA C1q status or titer at the time of first detection can independently predict allograft outcomes. In 70 renal transplant recipients who developed dn DSA we performed LABScreen single antigen bead assays using: neat, neat with ethylenediaminetetraacetic acid (EDTA), three dilutions after EDTA treatment (1:16, 1:64, 1:1024) and the C1q assay (neat sera). Histologic and clinical outcomes were correlated with the C1q assay or dn DSA titer. C1q positivity correlated with dn DSA titer (p dn DSA (p dn DSA development, but did not predict T-cell mediated rejection (TCMR) or antibody mediated rejection (ABMR). De novo DSA titer correlated with glomuerulitis, interstitial inflammation, tubulitis, peritubular capillaritis, and C4d scores, and predicted TCMR (p dnDSA development or other known predictors of post-dnDSA graft loss (i.e. clinical phenotype or non-adherence) neither C1q status nor dn DSA titer were independent predictors of a composite outcome of a 50% decline in eGFR or allograft loss (Fig. 1). Therefore, at present the additional cost and time associated with these approaches is not justified in routine practise. However, in cases where a renal biopsy is not possible dn DSA titer correlates with TCMR, and AMBR, and may assist in risk stratification. Download high-res image (239KB) Download full-size image

A new research programme involving eight Danish institutions is described. The programme started in 1993 and is expected to run for 5 years. The primary objective of the research initiative is to exploit and integrate the knowledge of several institutions and disciplines for the benefit of the production of rainbow trout. The programme includes several projects with aspects of disease prevention, genetics, and nutrition. In most of the projects, the work has been divided into stages of 2 and 3 years, respectively. During a 2 year period, production, management, and health status are recorded at the participating fish farms, and all data are organized in a database. Diseases cause major problems in rainbow trout production, therefore a great deal of the effort in this programme deals with diseases caused by vira, bacteria and parasites. On the basis of the database, epidemiological examinations are carried out as well as investigations of the possibilities of preventive measures and cost-benefit analyses. In the genetic studies, polymorphic genetic markers will be developed and used for analysis of the genetic structure of selected fish stocks. Microsatellites will be developed and introduced in the study. Primarily genetic differences between lines/strains and their crossings will be estimated with the purpose of describing the genetic level and the importance of additive and non-additive genetic effects. In the nutritional area the product quality and pollution questions will be in focus.

Elucidating how collagen fibril growth is regulated is important in determining how tissues are assembled. Fibrils are deposited as segments. The growth of these segments is an important determinant of tissue architecture, sta- bility, and mechanical attributes. Fibril segments were isolated from developing tendons and their structure characterized. The post-depositional changes leading to linear and lateral growth of fibrils also were examined. Segments extracted from 14-day chicken embryo tendons had a mean length of 29 pm. The segments were asymmetric, having a short and a long tapered end. Most of the segments were centrosymmetric with respect to molecular packing. Segments extracted from 12- to 16-day tendons had the same structure, but mean segment length increased incrementally due to the addition of an increasingly large population of longer segments. At 17 days of development there was a precipitous increase in segment length. The morphological data indicate that the increase in length was the result of lateral associ- ations among adjacent segments. Analysis demon- strated that this fibril growth was associated with a significant decrease in fibril associated decorin. Using immunoelectron microscopy, decorin was seen to decrease significantly at 18 days of devel- opment. When decorin content was biochemically determined, a decrease also was observed. Decorin mRNA also decreased relative to fibrillar collagen mRNA during the same period. These data support the hypothesis that a decrease in fibril-associated decorin is necessary for fibril growth associated with tissue maturation. Growth through post-depositional fusion allows for appo- sitional and intercalary growth and would be es- sential for normal development, growth, and repair. 0 1995 Wiley-Liss, Inc.

The interactions of small leucine-rich proteoglycans (SLRPs) with collagen fibrils, their association with water, and their role in fibrillogenesis suggests that SLRPs may play an important role in tendon mechanics. Some studies have assessed the role of SLRPs in the mechanical response of the tendon, but the relationships between sophisticated mechanics, assembly of collagen, and SLRPs have not been well characterized. Decorin content was varied in a dose dependent manner using decorin null, decorin heterozygote, and wild type mice. Quantitative measures of mechanical (tension and compression), compositional, and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was increased in the decorin heterozygous tendons compared to wild type. These tendons also had a significant decrease in total collagen and no structural changes compared to wild type. Decorin null tendons did not have any mechanical changes; however, a significant decrease in the average fibril diameter was found. No differences were seen between genotypes in elastic or compressive properties, and all tendons demonstrated viscoelastic mechanical dependence on strain rate and frequency. These results suggest that decorin, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis. In addition, reductions in decorin do not cause large changes in indentation compressive properties, suggesting that other factors contribute to these properties. Understanding these relationships may ultimately help guide development of tissue engineered constructs or treatment modalities.

During different stages in the development of the avian cornea, various collagen types have been shown to participate in matrix formation and have been implicated in morphogenesis. One of these is the fibril-associated collagen type IX. This molecule is present when the primary corneal stroma is in a compact state, but rapidly disappears just prior to stromal swelling and its invasion by mesenchymal cells. The temporospatial pattern of the disappearance of type IX collagen in the developing cornea suggests that this molecule may be involved in stabilizing the primary corneal stromal matrix by interacting either with other type IX collagen molecules or with other matrix components. To explore further whether the removal of type IX collagen is involved in stromal swelling, we have employed an in vitro culture system in which swelling of the primary stroma and mesenchymal cell invasion can be experimentally manipulated by culturing chick corneal explants on a Nuclepore filter support in the presence or absence of an associated lens. We have also examined the effect of exogenously added human recombinant tissue inhibitor of metalloproteinases (TIMP-1) on the presence of type IX collagen and cellular invasion. When stage 25—26+ corneal explants were cultured with an associated lens, the primary stroma did not swell; immunohistochemically detectable type IX collagen was still present, and mesenchymal cell invasion failed to occur. Conversely, when the same stages of corneal explants were cultured without an associated lens, the primary stroma swelled; type IX collagen disappeared, and mesenchymal cell migration occurred. Under both conditions, however, the type II collagen of the stroma, which is known to be a component of the striated fibrils, remained clearly detectable and with time even seemed to increase in amount. This result is consistent with the proposition that type IX collagen is one factor involved in maintaining the primary stroma as a compact matrix, possibly by functioning as a bridging/stabilizing factor. When TIMP was added to cultures of corneal explants, type IX collagen remained detectable in focal regions, suggesting that one or more metalloproteinases are involved in the removal of the type IX collagen. In addition, some of these type IX-containing regions contained mesenchymal cells, suggesting that in addition to type IX collagen other factors are likely to be involved in regulating mesenchymal cell migration. © 1994 Wiley-Liss, Inc.

Bowman's membrane is an acellular matrix of the cornea which lies between the epithelial basal lamina and the corneal stroma. By immunoelectron microscopy, we have determined that types I and V collagen are components of the collagen fibrils in Bowman's membrane of the chick cornea. Although these same components are found in the fibrils of the stroma, the fibrils of Bowman's membrane are smaller in diameter and less uniform than those of the stroma. At early stages of development, the corneal epithelium synthesizes the types I and II collagen of the primary stroma. We therefore asked whether it might also be capable of synthesizing the type V collagen found in Bowman's membrane at later stages of development. Our results, using competitive polymerase chain reaction to quantitate mRNA from avian corneal cells, indicate that the amount of alpha 1(V) collagen mRNA present in epithelia, relative to alpha 2(I) collagen mRNA, is greater than that in stromal fibroblasts. We postulate that this enables the epithelium to synthesize a higher ratio of type V to type I collagen than the stroma and that this proportionally higher amount of type V might account for the ultrastructural appearance of the fibrils in Bowman's membrane.

In situ hybridization is a technique with widespread application. However, its usefulness has been limited by the need for radioactive materials and the requirement for the DNA to be cloned onto an appropriate vector. We have utilized the polymerase chain reaction to directly incorporate a T7 RNA polymerase promoter sequence onto the cDNA for interleukin-2. Digoxigenin-labelled riboprobes were then synthesized using this PCR product as a template. The digoxigenin-labelled riboprobes were then used in non-radioactive in situ hybridization to detect messenger RNA for interleukin-2 in mitogen stimulated peripheral blood mononuclear cells. This methodology has the potential for widespread application in immunology and cytokine research.

Collagen V is a regulatory fibril-forming collagen that forms heterotypic fibrils with collagen I. Deletion of collagen V in the mouse is associated with a lack of fibril assembly in the embryonic mesenchyme, with a resultant lethal phenotype. The current work elucidates the regulatory roles of collagen V during development and growth of tissues. A conditional mouse model with a mutation in Col5a1 was developed using a Cre-loxP approach. Col5a1 was ablated in Col5a1flox/flox mice using a cornea stroma-specific Kera-Cre driver mouse to produce a bitransgenic Col5a1Δst/Δst line that is null for collagen V. This permits analyses of the corneal stroma, a widely used model for studies of collagen V. The collagen-V-knockout stroma demonstrated severe dysfunctional regulation of fibrillogenesis. Fibril diameters were significantly increased, with an abnormal, heterogeneous distribution; fibril structure was abnormal, fibril number was decreased and lamellae were disorganized with decreased stroma thickness. The phenotype was more severe in the anterior versus posterior stroma. Opacity was demonstrated throughout the Col5a1Δst/Δst stroma, with significantly increased haze intensity compared with control mice. These data indicate central regulatory roles for collagen V in fibril and matrix assembly during tissue development, with dysfunctional regulation resulting in a functional loss of transparency.

During neonatal development, tendons undergo a well orchestrated process whereby extensive structural and compositional changes occur in synchrony to produce a normal tissue. Conversely, during the repair response to injury, structural and compositional changes occur, but a mechanically inferior tendon is produced. As a result, developmental processes have been postulated as a potential paradigm for elucidation of mechanistic insight required to develop treatment modalities to improve adult tissue healing. The objective of this study was to compare and contrast normal development with injury during early and late developmental healing. Using backwards multiple linear regressions, quantitative and objective information was obtained into the structure–function relationships in tendon. Specifically, proteoglycans were shown to be significant predictors of modulus during early developmental healing but not during late developmental healing or normal development. Multiple independent parameters predicted percent relaxation during normal development, however, only biglycan and fibril diameter parameters predicted percent relaxation during early developmental healing. Lastly, multiple differential predictors were observed between early development and early developmental healing; however, no differential predictors were observed between late development and late developmental healing. This study presents a model through which objective analysis of how compositional and structural parameters that affect the development of mechanical parameters can be quantitatively measured. In addition, information from this study can be used to develop new treatment and therapies through which improved adult tendon healing can be obtained.

Autosomal-dominant congenital stromal corneal dystrophy (CSCD) is a human genetic disease characterized by corneal opacities beginning shortly after birth. It is linked to a frameshift mutation in decorin, resulting in a C-terminal truncation lacking 33 amino acids that includes the “ear” repeat, a feature specific for small leucine-rich proteoglycans. Our goals are to elucidate the roles of the mutant decorin in CSCD pathophysiology and to decipher the mechanism whereby mutant decorin affects matrix assembly. A novel animal model that recapitulates human CSCD was generated. This transgenic mouse model targets expression of truncated decorin to keratocytes, thereby mimicking the human frameshift mutation. Mutant mice expressed both wild-type and mutant decorin. Corneal opacities were found throughout, with increased severity toward the posterior stroma. The architecture of the lamellae was disrupted with relatively normal lamellae separated by regions of abnormal fibril organization. Within abnormal zones, the interfibrillar spacing and the fibril diameters were increased. Truncated decorin negatively affected the expression of endogenous decorin, biglycan, lumican, and keratocan and positively affected fibromodulin. Our results provide a mechanistic explanation for the generation of corneal opacities in CSCD. Thus, truncated decorin acts in a dominant-negative manner to interfere dually with matrix assembly and binding to receptor tyrosine kinases, thereby causing abnormal expression of endogenous small leucine-rich proteoglycans leading to structural abnormalities within the cornea and vision loss.

During neonatal development, tendons undergo a well orchestrated process whereby extensive structural and compositional changes occur in synchrony to produce a normal tissue. Conversely, during the repair response to injury, structural and compositional changes occur, but a mechanically inferior tendon is produced. As a result, developmental processes have been postulated as a potential paradigm through which improved adult tissue healing may occur. By examining injury at distinctly different stages of development, vital information can be obtained into the structure-function relationships in tendon. The mouse is an intriguing developmental model due to the availability of assays and genetically altered animals. However, it has not previously been used for mechanical analysis of healing tendon due to the small size and fragile nature of neonatal tendons. The objective of this study was to evaluate the differential healing response in tendon at two distinct stages of development through mechanical, compositional, and structural properties. To accomplish this, a new in vivo surgical model and mechanical analysis method for the neonatal mouse Achilles tendons were developed. We demonstrated that injury during early development has an accelerated healing response when compared to injury during late development. This accelerated healing model can be used in future mechanistic studies to elucidate the method for improved adult tendon healing.

Extracellular matrices are composed of collagens, proteoglycans, glycosaminoglycans, glycoproteins, and elastin. Extracellular matrix not only serves as structural scaffolds in organs and tissues, but also determines cellular function through cell–matrix interactions. Accordingly, the structures and organization of extracellular matrices are diverse and adapted to tissue-specific function. This chapter focuses on the collagen family. There are 28 different types of collagen that assemble into a variety of supramolecular structures including fibrils, microfibrils, and network-like structures. This chapter begins with a discussion of collagen molecules. This is followed by a definition of the supramolecular structure of different collagen types and their assembly and function within extracellular matrices. A discussion of general mechanistic principles involved in the assembly of collagen-containing suprastructures is presented. Finally, the regulation of tissue-specific collagen fibrillogenesis is used to illustrate how these general principles are applied in different tissues to generate the diversity in extracellular matrix structures and functions observed.

The Banff classification is ill suited to detect subtle histologic progression in renal allografts. We present image analysis methodology to precisely quantify IF in pediatric renal allograft biopsies routinely stained with MT. The mean area %IF was determined in 105 pediatric renal allograft biopsies. Associations between %IF or Banff ci scores and estimated GFR were determined using GEE modeling. Logistic regression was used to estimate IF progression. Percent IF (mean ± s.d.) was 6.83% ± 3.94, 10.39 ± 5.23%, and 20.53 ± 8.74 in patients with ci0, ci1, and ci2, respectively. The difference in %IF between biopsies with ci0, ci1, and ci2 was not proportionately incremental: compared to ci2, ci0 had 67% less IF (p0.0001), while ci1 had 48% less IF (p0.0001). AR had no impact on the precision of %IF measurements. Each 0.5% decrement in %IF was associated with a 1 mL/min per 1.73 m² increase in GFR (p0.004). Histologic progression was demonstrated by increasing %IF values (p0.0001) and could be estimated by IF = 2.61 × (months) + 6.43. This readily adaptable methodology may be used for the longitudinal assessment of IF in pediatric protocol renal allograft biopsies.

The collagens are a family of 28 extracellular matrix glycoproteins. Collagens are composed of 3 polypeptides, called alpha chains. Each α chain contains at least one stiff, rod-like domain of varying length with a glycine at every third residue that can form a triple helix, i.e., collagenous domain. Collagens V and XI are members of the fibril-forming class of collagens, along with collagens I, II, III, XXIV and XXVII. Collagens V and XI comprise a subclass of regulatory fibrillar collagens that co-assemble with collagens I, II and III. All collagens are trimers composed of 3 α chains with at least one triple helical or collagen (COL) domain. Alpha chains of collagens V (P20908 and P25940) and XI (P12107 and P13942) are encoded by genes with different phylogenetic origins, belonging to different clades or `ancestral groups' (clade 2) from collagens I, II and III (clade 1) or XXIV and XXVII (clade 3). Clade 1 also contains the α2(V) gene (P05997; i.e., the α2 chain of collagen V) and the α1(II) gene, termed α3(XI) when part of a collagen XI trimer (P02458). These genes code for multi-domain polypeptide chains with features common to all fibrillar collagens: a signal peptide; an N-terminal non-collagenous region (NC3); a short N-terminal triple helical domain of interrupted Gly-X–Y repeats (COL2), where X and Y can be any amino acid, but are often proline and hydroxyproline, respectively; a short non-collagenous domain (NC2 or `hinge'); the main triple helical or collagenous domain of continuous (GlyX–Y)n repeats (COL1); and a non-collagenous C-propeptide (NC1) (Fig.1).

The cornea consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells. The stroma consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25-30 nm fibrils that are regularly packed within lamellae and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sulfate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-beta would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.

During neonatal development, tendons undergo a well orchestrated process whereby extensive structural and compositional changes occur in synchrony to produce a normal tissue [1,2]. Similarly, during the repair response to injury, structural and compositional changes occur, but in this case, a mechanically inferior tendon is produced. As a result, the process of development has been postulated as a potential paradigm through which improved tissue healing may occur. By examining injury at distinctly different stages of development, we will obtain vital information into the structure-function relationships in tendon. Although the mouse is an intriguing model system due to the availability of assays and genetically altered animals, due to the small size and fragile nature of neonatal tendons, neonatal tendon injury has not been evaluated. Therefore, the objective of this study is to evaluate the differential healing response in neonatal tendon at two distinct stages of development. We hypothesized that when normalized, maximum stress and modulus will be significantly higher in early neonatal injury when compared to later neonatal injury. Also, when normalized, maximum stress, modulus and percent relaxation will be significantly increased over time in early neonatal injury but will remain low in later neonatal injury.Copyright © 2010 by ASME

Decorin (NM_133507) is a ubiquitous small extracellular proteoglycan. It is a composite molecule ~100kda in size with a protein core and attached GAGs. It was cloned from a human embryonic fibroblast line and named PG40 for its protein core, ~40Kda. It has been known as PG-S2, bone proteoglycans-II, small leucine-rich protein-1B, dermatan sulphate proteoglycan-II, but decorin (DCN) was adopted based on its association with collagen fibrils, i.e., it “decorated” fibrils. It is the prototype of an expanding family of small leucinerich repeat proteoglycans (SLRPs). The SLRP family has 5 classes and decorin is in class I.

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of C ol6a1−/− versus wild type tendons demonstrated disorganized extracellular micro-domains and associated collagen fibers in the C ol6a1−/− tendon. In C ol6a1−/− tendons, fibril structure and diameter distribution were abnormal compared to wild type controls. The diameter distributions were shifted significantly toward the smaller diameters in Col6a1−/− tendons compared to controls. An analysis of fibril density (number/μm 2 ) demonstrated a ~ 2.5 fold increase in the Col6a1−/− versus wild type tendons. In addition, the fibril arrangement and structure were aberrant in the peri-cellular regions of Col6a1−/− tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross-sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1−/− tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell–matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.

We previously showed an agarose overlay on keratocytes cultured in media containing pharmacological levels of insulin enhanced collagen processing and collagen fibril formation. In this study, we compared collagen processing by keratocytes cultured in media containing physiological levels of IGF-I, TGF-β, FGF-2, and PDGF in standard and in agarose overlay cultures. Pepsin digestion/SDS PAGE was used to determine the levels of procollagen secreted into the media and the collagen content of the ECM associated with the cell layer. Distribution of collagen type I and fibronectin in the ECM of the agarose cultures was determined by immunoflorescence. Collagen fibril and keratocyte morphology was evaluated by electron microscopy. The agarose overlay significantly enhanced the cell number in the IGF-I, TGF-β and PDGF treated cultures by 2–3 fold. The overlay also significantly enhanced the processing of procollagen to collagen fibrils from 29% in standard cultures to 63–68% in agarose cultures for the IGF-I and PDGF cultures, and from 66% in standard culture to 85% in agarose culture for the TGF-β cultures. Cell accumulation and collagen processing was not enhanced by agarose overlay of the FGF-2 treated cultures. Collagen type I and fibronectin were more uniformly distributed and the collagen fibrils smaller in the ECM of the TGF-β treated cultures. Keratocytes in the FGF-2 treated cultures were in close cell contact with few collagen fibrils while IGF-I, TGF-β, and PDGF cultures had an extensive ECM with abundant collagen fibrils. The results of this study indicate that the agarose overlay enhances collagen fibril assembly and cell accumulation by keratocytes when both collagen synthesis and cell proliferation are stimulated.

Corneal scarring from trauma and inflammation disrupts vision for millions worldwide, but corneal transplantation, the primary therapy for corneal blindness, is unavailable to many affected individuals. In this study, stem cells isolated from adult human corneal stroma were examined for the ability to correct stromal opacity in a murine model by direct injection of cells into the corneal stroma. In wild-type mice, injected human stem cells remained viable for months without fusing with host cells or eliciting an immune T-cell response. Human corneal-specific extracellular matrix, including the proteoglycans lumican and keratocan, accumulated in the treated corneas. Lumican-null mice have corneal opacity similar to that of scar tissue as a result of disruption of stromal collagen organization. After injection with human stromal stem cells, stromal thickness and collagen fibril defects in these mice were restored to that of normal mice. Corneal transparency in the treated mice was indistinguishable from that of wild-type mice. These results support the immune privilege of adult stem cells and the ability of stem cell therapy to regenerate tissue in a manner analogous to organogenesis and clearly different from that of normal wound healing. The results suggest that cell-based therapy can be an effective approach to treatment of human corneal blindness. Disclosure of potential conflicts of interest is found at the end of this article.

Decorin and biglycan are class I small leucine-rich proteoglycans (SLRPs) involved in regulation of collagen fibril and matrix assembly. We hypothesize that tissue-specific matrix assembly, such as in the cornea, requires a coordinate regulation involving multiple SLRPs. To this end, we investigated the expression of decorin and biglycan in the cornea of mice deficient in either SLRP gene and in double-mutant mice. Decorin and biglycan exhibited overlapping spatial expression patterns throughout the corneal stroma with differential temporal expression. Whereas decorin was expressed at relatively high levels in all developmental stages, biglycan expression was high early, decreased during development, and was present at very low levels in the mature cornea. Ultrastructural analyses demonstrated comparable fibril structure in the decorin- and biglycan-null corneas compared with wild-type controls. We found a compensatory up-regulation of biglycan gene expression in the decorin-deficient mice, but not the reverse. Notably, the corneas of compound decorin/biglycan-null mice showed severe disruption in fibril structure and organization, especially affecting the posterior corneal regions, corroborating the idea that biglycan compensates for the loss of decorin. Fibrillogenesis assays using recombinant decorin and biglycan confirmed a functional compensation, with both having similar effects at high SLRP/collagen ratios. However, at low ratios decorin was a more efficient regulator. The use of proteoglycan or protein core yielded comparable results. These findings provide firm genetic evidence for an interaction of decorin and biglycan during corneal development and further suggest that decorin has a primary role in regulating fibril assembly, a function that can be fine-tuned by biglycan during early development.

OBJECTIVE To determine the prevalence of macroalbuminuria and to describe the clinical and renal pathological changes associated with macroalbuminuria in a population of Canadian First Nation children and adolescents with type 2 diabetes. RESEARCH DESIGN AND METHODS We conducted a retrospective chart review at a single tertiary care pediatric diabetes center, and a case series was constructed. We collected data on microalbuminuria (≥3 mg/mmol creatinine [26.5 mg/g]) and macroalbuminuria (≥28 mg/mmol creatinine [247.5 mg/g]), estimated glomerular filtration rate, renal pathology, and aggravating risk factors (poor glycemic control, obesity, hypertension, glomerular hyperfiltration, hypercholesterolemia, smoking, and exposure to diabetes in utero). RESULTS We reviewed 90 charts of children and adolescents with type 2 diabetes. A total of 53% had at least one random urine albumin-to-creatinine ratio ≥3 mg/mmol (26.5 mg/g). There were 14 of 90 (16%) who had persistent macroalbuminuria at or within 8 years of diagnosis of diabetes. Of these 14 subjects, 1 had orthostatic albuminuria and 3 had spontaneous resolution of albuminuria. A total of 10 had renal biopsies performed. There were 9 of 10 who exhibited immune complex disease or glomerulosclerosis, and none had classic diabetic nephropathy. CONCLUSIONS This study suggests that the diagnosis of renal disease in children with type 2 diabetes cannot be reliably determined by clinical and laboratory findings alone. Renal biopsy is necessary for accurate diagnosis of renal disease in children and adolescents with type 2 diabetes and macroalbuminuria. The additional burden of nondiabetic kidney disease may explain the high rate of progression to end-stage kidney failure in this population.

On the basis of recent experimental results obtained using a model biological system, we argue that well-known physical phenomena like wetting and percolation may play a significant role in morphogenetic changes which occur during development. We also present results of scanning microscopy studies on the structure of the system, which support our theoretical conclusions.

Fibroblasts are responsible for the synthesis, assembly, deposition, and organization of extracellular matrix molecules, and thus determine the morphology of connective tissues. Deposition of matrix molecules occurs in extracellular compartments, where the sequential stages are under cellular control. Cell orientation/polarity is important in determining how the cell orients these extracytoplasmic compartments and therefore how the matrix is assembled and oriented. However, the control of cell orientation is not understood. Fibroblasts from three tissues with different morphologies were studied to determine whether cells maintained their characteristic phenotype. Fibroblasts from cornea, which in vivo are oriented in orthogonal layers along with their matrix; from tendon, a uniaxial connective tissue, where cells orient parallel to each other; and from dermis, a connective tissue with no apparent cellular orientation, were used to study cell morphology and orientation in three-dimensional collagen gels. The different cells were grown for 3 and 7 days in identical three-dimensional collagen gels with a nonoriented matrix. Confocal fluorescence microscopy demonstrated that corneal fibroblasts oriented perpendicular to one another at 3 days, and after 7 days in hydrated gels these cells formed orthogonal sheets. Tendon fibroblasts were shown by the same methods to orient parallel to one another in bundles at both 3 and 7 days, throughout the depth of the gel. Dermal fibroblasts showed no apparent orientation throughout the hydrated gels at either time point examined. The organization of these different cell types was consistent with their tissue of origin as was the cell structure and polarity. These studies imply that cellular and tissue phenotype is innate to differentiated fibroblasts and that these cells will orient in a tissue-specific manner regardless of the extracellular matrix present.

The role of matrix metalloproteinases (MMPs) in collagen fibrillogenesis during development has been studied in the well-characterized chicken metatarsal tendon. Collagen fibrils are initially assembled as intermediates, and the mature fibrils assemble by linear and lateral growth from these intermediates. We hypothesize that this involves the turnover of fibril-associated molecules mediated by the expression and activation of matrix metalloproteinase-2 (MMP-2). We demonstrate changes in the ratio of full-length to truncated MMP-2 during tendon development, consistent with enzyme activation. The level of full-length proMMP-2 remains relatively unchanged, although the truncated form of MMP-2 is highest prior to and during fibril growth. Membrane-type matrix metalloproteinase-3 (MT3-MMP, MMP-16) is fibroblast-associated and involved in the regulation of MMP-2 and in direct matrix turnover. The ratio of full-length proMT3-MMP/truncated (active) MT3-MMP has a pattern similar to that of full-length proMMP-2/truncated (active) MMP-2 during tendon development. Regulation of proMMP-2 activation involves complex formation with active MT3-MMP and TIMP-2. The constantly low TIMP-2 expression seen in tendon development is consistent with this role. Isolation of collagen fibrils from pre-fibril growth tendons (14 day) in the presence of activated MMP-2 is associated with premature fibril growth observed as increased fibril diameters compared with controls. These data implicate MMP-2/MT3-MMP in the initiation and progression of fibril growth, matrix assembly, and tendon development. This may involve the turnover of fibril-associated molecules involved in regulating linear and lateral growth, such as small leucine-rich proteoglycans and fibril-associated collagens. Activation of proMMP-2 dependent on MT3-MMP would allow the focal control of turnover.

'Fibroblast' is a generic term for a population of cells responsible for the establishment, maintenance and repair of three-dimensional form in multicellular organisms. These cells are a major, semi-permanent resident of the 'extracellular space' and the fibroblast compartmentalizes this space to various purposes during the formation of collagen fibrils and fibril bundles. The boundaries of these extracellular compartments blend and overlap, but discrete regions can be identified which are involved in collagen fibril assembly and fibril bundle assembly. The formation of these extracellular compartments in both the tendon and cornea results from a series of fusions of membrane-limited structures, beginning with collagen secretory vacuoles fusing with the cell surface to form deep surface recesses within which fibrils assemble; and progressing through the lateral fusion of these recesses with the consequent formation of fibril bundles. The topography of the fibroblast is also specialized for matrix anchorage, for matrix and cell repositioning, and for matrix degradation.

Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

The small-diameter fibrils of the chick corneal stroma are heterotypic, composed of both collagen types I and V. This tissue has a high concentration of type V collagen relative to other type I-containing tissues with larger-diameter fibrils, suggesting that heterotypic interactions may have a regulatory role in the control of fibril diameter. The interactions of collagen types I and V were studied using an in vitro self-assembly system. Collagens were purified from lathyritic chick embryos in the presence of protease inhibitors. The type V collagen preparations contained higher molecular weight forms of the alpha 1(V) and alpha 2(V) chains constituting 60–70% of the total. Rotary-shadow electron micrographs showed a persistence of a small, pepsin-sensitive terminal region in an amount consistent with that seen by electrophoresis. In vitro, this purified type V collagen formed thin fibrils with no apparent periodicity, while type I collagen fibrils had a broad distribution of large diameters. However, when type I collagen was mixed with increasing amounts of type V collagen a progressive and significant decrease in both the mean fibril diameter and the variance was observed for D periodic fibrils. The amino-terminal domain of the type V collagen molecule was required for this regulatory effect and in its absence little diameter reducing activity was observed. Electron microscopy using collagen type-specific monoclonal antibodies demonstrated that the fibrils formed were heterotypic, containing both collagen types I and V. These data indicate that the interaction of type V with type I collagen is one mechanism modulating fibril diameter and is at least partially responsible for the regulation of collagen fibril formation.

The organization of type IV collagen in the unconventional basement membrane of the corneal endothelium (Descemet's membrane) was investigated in developing chicken embryos using anti-collagen mAbs. Both immunofluorescence histochemistry and immunoelectron microscopy were performed. In mature embryos (greater than 15 d of development), the type IV collagen of Descemet's membrane was present as an array of discrete aggregates of amorphous material at the interface between Descemet's membrane and the posterior corneal stroma. Immunoreactivity for type IV collagen was also observed in the posterior corneal stroma as irregular plaques of material with a morphology similar to that of the Descemet's membrane-associated aggregates. This arrangement of Descemet's membrane-associated type IV collagen developed from a subendothelial mat of type IV collagen-containing material. This mat, in which type IV collagen-specific immunoreactivity was always discontinuous, first appeared at the time a confluent endothelium was established, well before the onset of Descemet's membrane formation. Immunoelectron microscopy of mature corneas revealed that the characteristic nodal matrix of Descemet's membrane itself was unreactive for type IV collagen, but was penetrated at intervals by projections of type IV collagen-containing material. These projections frequently appeared to contact cell processes from the underlying corneal endothelium. This spatial arrangement of type IV collagen suggests that it serves to suture the corneal endothelium/Descemet's membrane to the dense interfacial matrix of the posterior stroma.

Fibulin-2 is an extracellular matrix protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development. To examine its role in vivo, we generated mice that do not express the fibulin-2 gene (Fbln2) through homologous recombination of embryonic stem cells. Unexpectedly, the fibulin-2-null mice were viable and fertile and did not display gross and anatomical abnormalities. Histological and ultrastructural analyses revealed that elastic fibers assembled normally in the absence of fibulin-2. No compensatory up-regulation of mRNAs for other fibulin members was detected in the aorta and skin tissue. However, in the fibulin-2 null aortae, fibulin-1 immunostaining was increased in the inner elastic lamina, where fibulin-2 preferentially localizes. The results demonstrate that fibulin-2 is not required for mouse development and elastic fiber formation and suggest possible functional redundancy between fibulin-1 and fibulin-2.

To investigate whether targeted expression of lumican in the mouse cornea rescued the Lum(-/-) phenotype.Lum(-/-)/Kera-Lum mice were generated by crossing Lum(-/-) mice with Kera-Lum transgenic mice that overexpressed lumican under the control of the keratocan promoter. Mouse eyes were analyzed in vivo by confocal microscopy through focusing (CMTF) to determine corneal sublayer thickness and haze. Subsequently, one cornea from each mouse was processed for SDS-PAGE/western blotting while the other was used for either electron microscopy (EM) or real-time polymerase chain reaction (RT-PCR).Overall, corneas of Lum(-/-)/Kera-Lum mice showed significant improvement over Lum(-/-) but were still deficient when compared to wildtype (WT) mice. Specifically, analysis of Lum(-/-)/Kera-Lum mouse eyes by CMTF showed a similar stromal but slightly increased epithelial thickness compared to matching Lum(-/-) mice. Analysis of the CMTF scans for light backscattering revealed a small yet significant reduction in corneal haze in Lum(-/-)/Kera-Lum mice as compared to Lum(-/-) mice. At the EM level, the pronounced disarray of the posterior fibrillar matrix seen in Lum(-/-) mice was not observed in Lum(-/-)/Kera-Lum mice. Moreover, analyses of collagen fibril diameter distributions showed a significant reduction in the number of large-diameter (40 nm) fibrils in Lum(-/-)/Kera-Lum mice as compared to Lum(-/-) mice. No significant differences in keratocan expression were found at the mRNA level, but western blot analysis detected an approximately twofold increase in keratocan protein levels in Lum(-/-)/Kera-Lum over Lum(-/-) mice.Together these data suggest that despite the low keratocan promoter activity driving the transgene in Lum(-/-) cornea, transgenic lumican expression was sufficient to partially rescue corneal phenotypic deficiencies.

In 1999, our center implemented a policy of outpatient protocol biopsies as standard practice for the clinical management of pediatric renal allograft recipients. In order to determine the safety of this procedure, we conducted a retrospective chart audit of all outpatient renal allograft biopsies performed at our center. Biopsies were performed under conscious (midazolam) or procedural (propofol/fentanyl) sedation. Localization of the lower pole of the renal allograft was achieved with renal ultrasound. Using a Biopty gun with a 16-gauge needle, two cores were obtained. Patients were discharged four h post-biopsy. Patient demographics, hospital length of stay (LOS), specimen adequacy (per Banff criteria) and major and minor adverse events were recorded in a central database. Data were expressed as mean +/- SD. From June 1999 to July 2004, we performed 162 biopsies in 43 pediatric renal allograft recipients. Most patients underwent extraperitoneal transplantation (42/43, 97.7%) and were greater than five yr of age at biopsy (129/131 biopsies, 98.5%). The majority of these procedures (131/162, 80.9%) were conducted in the outpatient department, with 113 of 131 (86.3%) being obtained for protocol (n = 89) and one-month follow-up acute rejection therapy (n = 24) indications. Patients underwent 3.7 +/- 2.7 biopsies (range = 1-11). Specimen adequacy was achieved in 119 of 124 (96.0%) of documented cases. The overall incidence of adverse events was 12 of 131 (9.2%) biopsies, all of which were minor in severity. Macroscopic hematuria was the most common minor adverse event, occurring after 11 of 131 (8.4%) biopsies. While macroscopic hematuria prolonged LOS (adverse events vs. no adverse events: 23.0 +/- 26.0 vs. 8.6 +/- 4.1 h, p = 0), none of these episodes required major surgical or radiographic interventions. We conclude that in patients greater than five yr of age with extraperitoneal renal allografts, outpatient protocol biopsies using a 16-gauge needle are sufficiently safe to justify their inclusion in the routine clinical management of pediatric renal allograft recipients and in pediatric clinical trials.

The corneal endothelium is a monolayer of hexagonal cells derived from the neural crest that is responsible for maintaining the appropriate degree of corneal hydration necessary for vision. The normal endothelial cell count is 2 to 5 × 103 cells/mm2 in humans.1 An endothelial cell count below a threshold of 300 to 500 cells per mm2 is associated with irreversible corneal swelling, epithelial blistering, loss of vision, and need of transplantation.1,2 The current dogma is that mature corneal endothelial cells are quiescent, arrested in the G1-phase of the cell cycle, with limited proliferative capacity in vivo.3–5 Cell contact inhibition has been proposed to have a major role inhibiting endothelial replication.3 However, corneal endothelial cells can replicate in cell cultures and tissue explants. In addition, the presence of endothelial cells expressing progenitor cell markers in the human corneal periphery suggest that endothelial cells or at least some endothelial cells may replicate or have the ability to replicate in an appropriate environment.6–8 Two different facts strongly suggest that human endothelial cells proliferate in vivo and repopulate donor grafts. First, studies in sex-mismatched transplanted corneas showed the presence of a mixture of donor and recipient endothelial cells in the transplanted graft.9,10 Endothelial replacement by the host cells in failed edematous corneas, and also in cases where corneas were clear was demonstrated suggesting that endothelial replacement occurs in failed and successful transplants.9 Second, anecdotal clinical reports show that human endothelial cells can replicate in vivo in the periphery of the posterior cornea and migrate and restore Descemet's membrane, and the endothelial monolayer integrity reestablishing corneal transparency.11,12 Nestin, a class VI intermediate filament, has been used to identify cells with progenitor cell properties in the developing neural cortex,13 cultured primary neurons from the central and peripheral nervous systems,14 immortalized cell lines, and tumors.15 It is well documented that expression of nestin reflects the undifferentiated state of neural progenitor cells16 and that downregulation of nestin reactivity is associated with the differentiation of neural progenitor cells in the developing nervous system.16 Corneal keratocytes and endothelial cells originate from multipotent cranial neural crest cells that migrate rostrally to form facial structures.17 The low-affinity nerve growth factor receptor (NGFR) is one of the two receptor types for the neurotrophins and has been used as a neural crest stem cell marker.18–20 A pure or enriched population of neural crest stem cells has been isolated from mouse trunk neural tubes and embryonic peripheral nerves using low-affinity NGFR as a marker.18,19 These cells that are NGFR-positive had self-renewal capacity, and displayed multipotent differentiation properties. Other progenitor cell markers include Sox9 and leucine-rich repeat containing G protein-coupled receptor 5 (LGR5). Sex-determining region Y–box containing gene 9 (Sox9), high-mobility group box (Sox) transcription factors, is mutated in campomelic dysplasia, a disorder characterized by skeletal malformations, XY sex reversal, and neonatal lethality.21 Sex-determining region Y–box 9 is expressed during embryogenesis in several tissues and organs, including the central nervous system.22 Initially identified as a Wnt target gene in human colon cancer cell lines harboring Wnt-activating mutations,23 Lgr5 is a well-established epithelial intestinal stem cell marker.24,25 Detection of Ki-67 is commonly used in histological specimens to detect cell proliferation and provides an index of proliferation with prognostic value.26 It was initially reported in the early 1980s.27 It is a nonhistone nuclear and nucleolar protein strictly associated with cell proliferation and is detected during all the active phases of the cell cycle, but is absent in resting cells. Its strict association with cell proliferation and its coexpression with other well-known markers of proliferation indicate a pivotal role in cell division. Expression of Ki-67 is upregulated in the G2, M, and latter half of the S phase of the cell cycle.28 Progenitor cells are able to differentiate into one or more cell types, but do not have unlimited differentiation or replication properties like stem cells. Progenitor cells can adjust their cell-cycle properties, in homeostasis or during injury, to the circumstances that surround them.29 A classic way to search for stem cells or progenitors cell niches is the use of labeling agents like 5-bromo-2′-deoxyuridine (BrdU).29 Slow-dividing cells found in hair follicles and the corneal limbus have been previously described as stem cells.30 Therefore, this slow-cycling characteristic is considered a common feature to identify stem cells in different tissues and organs. Here we demonstrate the presence of populations of progenitor cells in the corneal periphery as well as the existence of slow-cycling cells in the extreme periphery of the corneal endothelium.

PURPOSE. To determine the effect of latanoprost on the expression of human matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the ciliary body. METHODS. Total RNA was isolated, and qualitative RT-PCR was performed to detect the mRNA of MMPs and TIMPs in human ciliary body tissue and explant cultures of ciliary body smooth muscle (CBSM) cells. CBSM cell cultures were treated with vehicle control or latanoprost acid for 24 hours. Quantitative RT-PCR of cell cultures from five different donors was performed to determine relative changes in expression. GAPDH served as an endogenous control. RESULTS. The mRNA of MMP-1, -2, -3, -11, -12, -14, -15, -16, -17, -19, and -24 as well as TIMP-1 to -4 were found in ciliary body tissue and CBSM cells. MMP-9 was present after latanoprost treatment. In control CBSM cells, the relative expression of MMP mRNA was MMP-2 and -14 MMP-24 MMP-1, -11, -15, -16, and -19 MMP-3 and 17, MMP-12. The relative expression of TIMP mRNA was TIMP-2 TIMP-1 TIMP-3 TIMP-4. Latanoprost increased MMP-3 (in three of five cultures), MMP-17 (in four of five cultures), and TIMP-3 (in all five cultures); MMP-1, -2, -12, -14, -15, and -16 and TIMP-4 were downregulated. CONCLUSIONS. The transcription of the genes for MMP-3 and -17 is increased by latanoprost treatment. MMP-9 is present after latanoprost treatment and may also mediate ECM changes. TIMP-3 is upregulated and may compensate for the increase in MMPs. These coordinated changes could be expected to mediate the latanoprost-induced alteration of ECM in the ciliary body. (Invest Ophthalmol Vis Sci. 2006;47:953‐963) DOI

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proalpha1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.

PURPOSE Type V collagen forms heterotypic fibrils with type I collagen and accounts for 10% to 20% of corneal collagen. The purpose of this study was to define the ocular phenotype resulting from mutations in the type V collagen genes COL5A1 and COL5A2 and to study the pathogenesis of anomalies in a Col5a1-deficient mouse. METHODS Seven patients with classic Ehlers-Danlos syndrome (EDS) due to COL5A1 haploinsufficiency and one with an exon-skipping mutation in COL5A2 underwent an ocular examination, corneal topography, pachymetry, and specular microscopy. A Col5a1-haploinsufficient mouse model of classic EDS was used for biochemical and immunochemical analyses of corneas. Light and electron microscopy were used to quantify stromal thickness, fibril density, fibril structure, and diameter. RESULTS Five males and three females (mean age, 26 +/- 13.57 years; range, 11-52) were studied. All patients had "floppy eyelids." The corneas of all eyes were thinner (mean corneal thickness: 435.75 +/- 12.51 microm) when compared with control corneas (568.89 +/- 28.46 microm; P < 0.0001). In the Col5a1+/- mouse cornea, type V collagen content was reduced by approximately 49%, and stromal thickness was reduced by approximately 26%. Total collagen deposition in Col5a1(+/-) corneas also was reduced. Collagen fibril diameters were increased, but fibril density was decreased throughout the stroma at all developmental stages. CONCLUSIONS In the eye, COL5A1 and COL5A2 mutations manifest as abnormally thin and steep corneas with floppy eyelids. Mechanisms involved in producing the latter anomalies probably involve altered regulation of collagen fibrillogenesis due to abnormalities in heterotypic type I/V collagen interactions similar to those observed in the Col5a1+/- mouse cornea.

Procollagen C proteinases (pCPs) cleave type I to III procollagen C propeptides as a necessary step in assembling the major fibrous components of vertebrate extracellular matrix. The protein PCOLCE1 (procollagen C proteinase enhancer 1) is not a proteinase but can enhance the activity of pCPs approximately 10-fold in vitro and has reported roles in inhibiting other proteinases and in growth control. Here we have generated mice with null alleles of the PCOLCE1 gene, Pcolce, to ascertain in vivo roles. Although Pcolce-/- mice are viable and fertile, Pcolce-/- male, but not female, long bones are more massive and have altered geometries that increase resistance to loading, compared to wild type. Mechanical testing indicated inferior material properties of Pcolce-/- male long bone, apparently compensated for by the adaptive changes in bone geometry. Male and female Pcolce-/- vertebrae both appeared to compensate for inferior material properties with thickened and more numerous trabeculae and had a uniquely altered morphology in deposited mineral. Ultrastructurally, Pcolce-/- mice had profoundly abnormal collagen fibrils in both mineralized and nonmineralized tissues. In Pcolce-/- tendon, 100% of collagen fibrils had deranged morphologies, indicating marked functional effects in this tissue. Thus, PCOLCE1 is an important determinant of bone mechanical properties and geometry and of collagen fibril morphology in mammals, and the human PCOLCE1 gene is identified as a candidate for phenotypes with defects in such attributes in humans.

Lumican is an extracellular matrix glycoprotein widely distributed in mammalian connective tissues. Corneal lumican modified with keratan sulfate constitutes one of the major proteoglycans of the stroma. Lumican-null mice exhibit altered collagen fibril organization and loss of corneal transparency. A closely related protein, keratocan, carries the remaining keratan sulfate of the cornea, but keratocan-null mice exhibit a less severe corneal phenotype. In the current study, we examined the effect of lumican overexpression in corneas of wild type mice. These mice showed no alteration in collagen organization or transparency but had increased keratocan expression at both protein and mRNA levels. Corneas of lumican-null mice showed decreased keratocan. This coupling of keratocan expression with lumican also was observed after intrastromal injection of a lumican expression minigene into the corneal stroma of Lum-/- mice. Small interfering RNA knockdown of lumican in vitro reduced keratocan expression, whereas co-injection of a lumican-expressing minigene with a beta-galactosidase reporter driven by the keratocan promoter demonstrated an increase of keratocan transcriptional activity in response to lumican expression in Lum-/- corneas in vivo. These observations demonstrate that lumican has a novel regulatory role in keratocan expression at the transcriptional level. Such results help provide an explanation for the differences in severity of corneal manifestation found in Lum-/- and Kera-/- mice. The results also suggest a critical level of small proteoglycans to be essential for collagen organization but that overabundance is not detrimental to extracellular matrix morphogenesis.

In the tendon, the development of mature mechanical properties is dependent on the assembly of a tendon-specific extracellular matrix. This matrix is synthesized by the tendon fibroblasts and composed of collagen fibrils organized as fibers, as well as fibril-associated collagenous and non-collagenous proteins. All of these components are integrated, during development and growth, to form a functional tissue. During tendon development, collagen fibrillogenesis and matrix assembly progress through multiple steps where each step is regulated independently, culminating in a structurally and functionally mature tissue. Collagen fibrillogenesis occurs in a series of extracellular compartments where fibril intermediates are assembled and mature fibrils grow through a process of post-depositional fusion of the intermediates. Linear and lateral fibril growth occurs after the immature fibril intermediates are incorporated into fibers. The processes are regulated by interactions of extracellular macromolecules with the fibrils. Interactions with quantitatively minor fibrillar collagens, fibril-associated collagens and proteoglycans influence different steps in fibrillogenesis and the extracellular microdomains provide a mechanism for the tendon fibroblasts to regulate these extracellular interactions.

In a small proportion of patients with an Ullrich-like clinical phenotype no mutations in the collagen VI genes are detected. Here we report on a novel muscle and tendon collagenopathy caused by mutations in COL12A1. In a consanguineous family with hypotonia, weakness and significant joint hyperlaxity we identified a homozygous frame shifting mutation in COL12A1 in the two affected children, causing complete absence of collagen XII in muscle and fibroblasts. Patients were unable to walk and developed scoliosis and respiratory insufficiency. In the second family the proband presented with milder hypotonia, motor delay, hyperlaxity and incomplete deficiency of collagen XII. We identified a de novo, presumably dominantly acting missense mutation. COL12A1 encodes collagen XII. Collagen XII is a homotrimer found in association with collagen I and acts as a cross-bridge between collagen fibrils while also interacting with tenascin X (TNX), heparin and decorin. We next investigated the muscle phenotype in a Col12a1 KO mouse, which showed age dependent weakness. At 9 months of age there was no significant difference in muscle fiber size, but the overall weight of the muscles was less with an altered slow-to-fast fiber type transition in both soleus and tibilias anterior muscles. Physiological measurements on isolated EDL showed protection from eccentric force drop and a decrease in passive force, suggesting an increased compliance of the matrix as well as decreased lateral and longitudinal force transmission to the matrix and tendon. We hypothesize that the functional abnormalities seen in muscle force measurement in COL12 KO mice originate at least partly from an underlying tendon and matrix pathology caused by the absence of collagen XII.