Proteoglycans of basement membranes: Crucial controllers of angiogenesis, neurogenesis, and autophagy.

Anti-angiogenic therapy is an established method for the treatment of several cancers and vascular-related diseases. Most of the agents employed target the vascular endothelial growth factor A, the major cytokine stimulating angiogenesis. However, the efficacy of these treatments is limited by the onset of drug resistance. Therefore, it is of fundamental importance to better understand the mechanisms that regulate angiogenesis and the microenvironmental cues that play significant role and influence patient treatment and outcome. In this context, here we review the importance of the three basement membrane heparan sulfate proteoglycans (HSPGs), namely perlecan, agrin and collagen XVIII. These HSPGs are abundantly expressed in the vasculature and, due to their complex molecular architecture, they interact with multiple endothelial cell receptors, deeply affecting their function. Under normal conditions, these proteoglycans exert pro-angiogenic functions. However, in pathological conditions such as cancer and inflammation, extracellular matrix remodeling leads to the degradation of these large precursor molecules and the liberation of bioactive processed fragments displaying potent angiostatic activity. These unexpected functions have been demonstrated for the C-terminal fragments of perlecan and collagen XVIII, endorepellin and endostatin. These bioactive fragments can also induce autophagy in vascular endothelial cells which contributes to angiostasis. Overall, basement membrane proteoglycans deeply affect angiogenesis counterbalancing pro-angiogenic signals during tumor progression, and represent possible means to develop new prognostic biomarkers and novel therapeutic approaches for the treatment of solid tumors.

CNS/PNS proteoglycans functionalize neuronal and astrocyte niche microenvironments optimizing cellular activity by preserving membrane polarization dynamics, ionic microenvironments, ion fluxes, neuronal activation, and network neurotransductive capacity.

Central and peripheral nervous system (CNS/PNS) proteoglycans (PGs) have diverse functional roles, this study examined how these control cellular behavior and tissue function. The CNS/PNS extracellular matrix (ECM) is a dynamic, responsive, highly interactive, space-filling, cell supportive, stabilizing structure maintaining tissue compartments, ionic microenvironments, and microgradients that regulate neuronal activity and maintain the neuron in an optimal ionic microenvironment. The CNS/PNS contains a high glycosaminoglycan content (60% hyaluronan, HA) and a diverse range of stabilizing PGs. Immobilization of HA in brain tissues by HA interactive hyalectan PGs preserves tissue hydration and neuronal activity, a paucity of HA in brain tissues results in a pro-convulsant epileptic phenotype. Diverse CS, KS, and HSPGs stabilize the blood-brain barrier and neurovascular unit, provide smart gel neurotransmitter neuron vesicle storage and delivery, organize the neuromuscular junction basement membrane, and provide motor neuron synaptic plasticity, and photoreceptor and neuron synaptic functions. PG-HA networks maintain ionic fluxes and microgradients and tissue compartments that contribute to membrane polarization dynamics essential to neuronal activation and neurotransduction. Hyalectans form neuroprotective perineuronal nets contributing to synaptic plasticity, memory, and cognitive learning. Sialoglycoprotein associated with cones and rods (SPACRCAN), an HA binding CSPG, stabilizes the inter-photoreceptor ECM. HSPGs pikachurin and eyes shut stabilize the photoreceptor synapse aiding in phototransduction and neurotransduction with retinal bipolar neurons crucial to visual acuity. This is achieved through Laminin G motifs in pikachurin, eyes shut, and neurexins that interact with the dystroglycan-cytoskeleton-ECM-stabilizing synaptic interconnections, neuronal interactive specificity, and co-ordination of regulatory action potentials in neural networks.

Microvascular damage, neuroinflammation and extracellular matrix remodeling in Col18a1 knockout mice as a model for early cerebral small vessel disease.

Collagen type XVIII (COL18) is an abundant heparan sulfate proteoglycan in vascular basement membranes. Here, we asked (i) if the loss of COL18 would result in blood-brain barrier (BBB) breakdown, pathological alterations of small arteries and capillaries and neuroinflammation as found in cerebral small vessel disease (CSVD) and (ii) if such changes may be associated with remodeling of synapses and neural extracellular matrix (ECM). We found that 5-month-old Col18a1-/- mice had elevated BBB permeability for mouse IgG in the deep gray matter, and intravascular erythrocyte accumulations were observed brain-wide in capillaries and arterioles. BBB permeability increased with age and affected cortical regions and the hippocampus in 12-month-old Col18a1-/- mice. None of the Col18a1-/- mice displayed hallmarks of advanced CSVD, such as hemorrhages, and did not show perivascular space enlargement. Col18a1 deficiency-induced BBB leakage was accompanied by activation of microglia and astrocytes, a loss of aggrecan in the ECM of perineuronal nets associated with fast-spiking inhibitory interneurons and accumulation of the perisynaptic ECM proteoglycan brevican and the microglial complement protein C1q at excitatory synapses. As the pathway underlying these regulations, we found increased signaling through the TGF-ß1/Smad3/TIMP-3 cascade. We verified the pivotal role of COL18 for small vessel wall structure in CSVD by demonstrating the protein's involvement in vascular remodeling in autopsy brains from patients with cerebral hypertensive arteriopathy. Our study highlights an association between the alterations of perivascular ECM, extracellular proteolysis, and perineuronal/perisynaptic ECM, as a possible substrate of synaptic and cognitive alterations in CSVD.

Endostatin in Renal and Cardiovascular Diseases.

Endostatin, a protein derived from the cleavage of collagen XVIII by the action of proteases, is an endogenous inhibitor known for its ability to inhibit proliferation and migration of endothelial cells, angiogenesis, and tumor growth. Angiogenesis is defined as the formation of new blood vessels from pre-existing vasculature, which is crucial in many physiological processes, such as embryogenesis, tissue regeneration, and neoplasia. SUMMARY: Increasing evidence shows that dysregulation of angiogenesis is crucial for the pathogenesis of renal and cardiovascular diseases. Endostatin plays a pivotal role in the regulation of angiogenesis. Recent studies have provided evidence that circulating endostatin increases significantly in patients with kidney and heart failure and may also contribute to disease progression. KEY MESSAGE: In the current review, we summarize the latest findings on preclinical and clinical studies analyzing the impact of endostatin on renal and cardiovascular diseases.

Deciphering the focal role of endostatin in Alzheimer's disease.

Alzheimer's disease (AD) is a paramount chronic neurodegenerative condition that has been affecting elderly people since the 1900s. It causes memory loss, disorientation, and poor mental function. AD is considered to be one of the most serious problems that dementia sufferers face. Despite extensive investigation, the pathological origin of Alzheimer's disease remains a mystery. The amyloid cascade theory and the vascular hypothesis, which stresses the buildup of Aß plaques, have dominated research into dementia and aging throughout history. However, research into this task failed to yield the long-awaited therapeutic miracle lead for Alzheimer's disease. Perhaps a hypothetical fragility in the context of Alzheimer's disease was regarded as a state distinct from aging in general, as suggested by the angiogenesis hypothesis, which suggests that old age is one state associated with upregulation of angiogenic growth factors, resulting in decreased microcirculation throughout the body. There has also been evidence that by controlling or inhibiting the components involved in the sequence of events that cause angiogenesis, there is a visible progression in AD patients. In Alzheimer's disease, one such antiangiogenic drug is being used.

Exploring the molecular role of endostatin in diabetic neuropathy.

For over a decade, diabetic neuropathy has exhibited great emergence in diabetic patients. Though there are numerous impediments in understanding the underlying pathology it is not that enough to conclude. Initially, there was no intricate protocol for diagnosis as its symptoms mimic most of the neurodegenerative disorders and demyelinating diseases. Continuous research on this, reveals many pathological correlates which are also detectable clinically. The most important pathologic manifestation is imbalanced angiogenesis/neo-vascularization. This review is completely focused on established pathogenesis and anti-angiogenic agents which are physiological signal molecules by the origin. Those agents can also be used externally to inhibit those pathogenic pathways. Pathologically DN demonstrates the misbalanced expression of many knotty factors like VEGF, FGF2, TGFb, NF-kb, TNF-a, MMP, TIMP, and many minor factors. Their pathway towards the incidence of DN is quite interrelated. Many anti-angiogenic agents inhibit neovascularization to many extents, but out of them predominantly inhibition of angiogenic activity is shared by endostatin which is now in clinical trial phase II. It inhibits almost all angiogenic factors and it is possible because they share interrelated pathogenesis towards imbalanced angiogenesis. Endostatin is a physiological signal molecule produced by the proteolytic cleavage of collagen XVIII. It has also a broad research profile in the field of medical research and further investigation can show promising therapeutic effects for benefit of mankind.

Lack of collagen XVIII leads to lipodystrophy and perturbs hepatic glucose and lipid homeostasis.

KEY POINTS: Extracellular matrix is highly remodelled in obesity and associates with the development of metabolic disorders, such as insulin resistance. Previously, we have shown that the lack of specific collagen XVIII isoforms impairs adipocyte differentiation in mice. Here, we show that mice lacking the medium and long isoforms of collagen XVIII develop insulin resistance and glucose intolerance and show elevated serum triglycerides and fat accumulation in the liver. We report that collagen XVIII-deficient mice have increased heat production at low temperatures. These results reveal a new role for collagen XVIII in the regulation of glucose and lipid metabolism, and they expand the understanding of the development of metabolic disorders. ABSTRACT: Liver and adipose tissues play important roles in the regulation of systemic glucose and lipid metabolism. Extracellular matrix synthesis and remodelling are significantly altered in these tissues in obesity and type 2 diabetes. Collagen XVIII is a ubiquitous extracellular matrix component, and it occurs in three isoforms which differ in terms of molecular size, domain structure and tissue distribution. We recently showed that, in mice, the lack of collagen XVIII, and especially its medium and long isoforms, leads to reduced adiposity and dyslipidaemia. To address the metabolic consequences of these intriguing observations, we assessed whole-body glucose homeostasis in mice challenged with a high-fat diet and in normal physiological conditions. We observed that, in the high caloric diet, the overall adiposity was decreased by 30%, serum triglyceride values were threefold higher and the steatotic area in liver was twofold larger in collagen XVIII knockout mice compared with controls. We demonstrated that mice lacking either all three collagen XVIII isoforms, or specifically, the medium and long isoforms develop insulin resistance and glucose intolerance. Furthermore, we found that ablation of collagen XVIII leads to increased heat production in low temperatures and to reduction of the high blood triglyceride levels of the knockout mice to the level of wild-type mice. Our data indicate that collagen XVIII plays a role in the regulation of glucose tolerance, insulin sensitivity and lipid homeostasis, principally through its ability to regulate the expansion of the adipose tissue. These findings advance the understanding of metabolic disorders.

Comment on: "Wnt Binding Affinity Prediction for Putative Frizzled-Type Cysteine-Rich Domains".

Dear Editor, [...].

Collagen XVIII Deposition in the Basement Membrane Zone beneath the Newly Forming Epidermis during Wound Healing in Mice.

The basement membrane (BM) is composed of various extracellular molecules and regulates tissue regeneration and maintenance. Here, we demonstrate that collagen XVIII was spatiotemporally expressed in the BM during skin wound healing in a mouse excisional wound-splinting model. Re-epithelialization was detected at days 3 and 6 post-wounding. The ultrastructure of epidermal BM was discontinuous at day 3, whereas on day 6 a continuous BM was observed in the region proximal to the wound edge. Immunohistochemistry demonstrated that collagen XVIII was deposited in the BM zone beneath newly forming epidermis in day 3 and 6 wounds. Laminin-332, known to be the earliest BM component appearing in wounds, was colocalized with collagen XVIII in the epidermal BM zone at days 3 and 6. The deposition of α1(IV) collagen and nidogen-1 in the epidermal BM zone occurred later than that of collagen XVIII. We also observed the short isoform of collagen XVIII in the epidermal BM zone at day 3 post-wounding. Collectively, our results suggested that collagen XVIII plays a role in the formation of the dermal-epidermal junction during re-epithelialization, and that it is the short isoform that is involved in the early phase of re-epithelialization.

Proteoglycans Are Attractive Biomarkers and Therapeutic Targets in Hepatocellular Carcinoma.

Proteoglycans, which consist of a protein core and glycosaminoglycan chains, are major components of the extracellular matrix and play physiological roles in maintaining tissue homeostasis. In the carcinogenic tissue microenvironment, proteoglycan expression changes dramatically. Altered proteoglycan expression on tumor and stromal cells affects cancer cell signaling pathways, which alters growth, migration, and angiogenesis and could facilitate tumorigenesis. This dysregulation of proteoglycans has been implicated in the pathogenesis of diseases such as hepatocellular carcinoma (HCC) and the underlying mechanism has been studied extensively. This review summarizes the current knowledge of the roles of proteoglycans in the genesis and progression of HCC. It focuses on well-investigated proteoglycans such as serglycin, syndecan-1, glypican 3, agrin, collagen XVIII/endostatin, versican, and decorin, with particular emphasis on the potential of these factors as biomarkers and therapeutic targets in HCC regarding the future perspective of precision medicine toward the "cure of HCC".

Endostatin: a promising biomarker in the cardiovascular continuum?

The current review article aims to provide an up-to-date summary of previous studies in humans that have reported the association between circulating endostatin levels and different cardiovascular phenotypes. We also aim to provide suggestions for future directions of future research evaluating endostatin as a clinically relevant cardiovascular biomarker. With a few exceptions, higher circulating levels of endostatin seem to reflect vascular and myocardial damage, and a worsened prognosis for cardiovascular events or mortality in individuals with hypertension, diabetes, kidney disease, cardiovascular disease, as well as in the general population. Circulating endostatin seems to be a promising biomarker for cardiovascular pathology, but there is not enough evidence to date to support the use of endostatin measurements in clinical practice.

An extracellular proteasome releases endostatin from human collagen XVIII.

Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.

Endostatin and transglutaminase 2 are involved in fibrosis of the aging kidney.

Endostatin (EST), an antiangiogenic factor, is enriched in aging kidneys. EST is also an interactive partner of transglutaminase 2 (TG2), an enzyme that cross-links extracellular matrix proteins. Here we tested whether EST and TG2 play a role in the fibrosis of aging. In wild-type mice, aging kidneys exhibited a 2- to 4-fold increase in TG2 paralleled by increased cross-linked extracellular matrix proteins and fibrosis. Mice transgenic to express EST showed renal fibrosis at a young age. One-month delivery of EST via minipumps to young mice showed increased renal fibrosis that became more robust when superimposed on folic acid-induced nephropathy. Upregulated TG2 and impaired renal function were apparent with EST delivery combined with folic acid-induced nephropathy. Subcapsular injection of TG2 and/or EST into kidneys of young mice not only induced interstitial fibrosis, but also increased the proportion of senescent cells. Thus, kidney fibrosis in aging may represent a natural outcome of upregulated EST and TG2, but more likely it appears to be a result of cumulative stresses occurring on the background of synergistically acting geronic (aging) proteins, EST and TG2.

Absence of collagen XVIII in mice causes age-related insufficiency in retinal pigment epithelium proteostasis.

Collagen XVIII has the structural properties of both collagen and proteoglycan. It has been found at the basement membrane/stromal interface where it is thought to mediate their attachment. Endostatin, a proteolytic fragment from collagen XVIII C-terminal end has been reported to possess anti-angiogenic properties. Age-related vision loss in collagen XVIII mutant mice has been accompanied with a pathological accumulation of deposits under the retinal pigment epithelium (RPE). We have recently demonstrated that impaired proteasomal and autophagy clearance are associated with the pathogenesis of age-related macular degeneration. This study examined the staining levels of proteasomal and autophagy markers in the RPE of different ages of the Col18a1 (-/-) mice. Eyes from 3, 6-7, 10-13 and 18 months old mice were enucleated and embedded in paraffin according to the routine protocol. Sequential 5 µm-thick parasagittal samples were immunostained for proteasome and autophagy markers ubiquitin (ub), SQSTM1/p62 and beclin-1. The levels of immunopositivity in the RPE cells were evaluated by confocal microscopy. Collagen XVIII knock-out mice had undergone age-related RPE degeneration accompanied by an accumulation of drusen-like deposits. Ub protein conjugate staining was prominent in both RPE cytoplasm and extracellular space whereas SQSTM1/p62 and beclin-1 stainings were clearly present in the basal part of RPE cell cytoplasm in the Col18a1 (-/-) mice. SQSTM1/p62 displayed mild extracellular space staining. Disturbed proteostasis regulated by collagen XVIII might be responsible for the RPE degeneration, increased protein aggregation, ultimately leading to choroidal neovascularization.

Collagens XV and XVIII show different expression and localisation in cutaneous squamous cell carcinoma: type XV appears in tumor stroma, while XVIII becomes upregulated in tumor cells and lost from microvessels.

As the second most common skin malignancy, cutaneous squamous cell carcinoma (cSCC) is an increasing health concern, while its pathogenesis at molecular level remains largely unknown. We studied the expression and localisation of two homologous basement membrane (BM) collagens, types XV and XVIII, at different stages of cSCC. These collagens are involved in angiogenesis and tumorigenesis, but their role in cancer development is incompletely understood. Quantitative RT-PCR analysis revealed upregulation of collagen XVIII, but not collagen XV, in primary cSCC cells in comparison with normal human epidermal keratinocytes. In addition, the Ha-ras-transformed invasive cell line II-4 expressed high levels of collagen XVIII mRNA, indicating upregulation in the course of malignant transformation. Immunohistochemical analyses of a large human tissue microarray material showed that collagen XVIII is expressed by tumor cells from grade 1 onwards, while keratinocytes in normal skin and in premalignant lesions showed negative staining for it. Collagen XV appeared instead as deposits in the tumor stroma. Our findings in human cSCCs and in mouse cSCCs from the DMBA-TPA skin carcinogenesis model showed that collagen XVIII, but not collagen XV or the BM markers collagen IV or laminin, was selectively reduced in the tumor vasculature, and this decrease associated significantly with cancer progression. Our results demonstrate that collagens XV and XVIII are expressed in different sites of cSCC and may contribute in a distinct manner to processes related to cSCC tumorigenesis, identifying these collagens as potential biomarkers in the disease.

Possible role of endostatin in the antiangiogenic therapy of diabetic retinopathy.

Diabetic retinopathy is one of various complications of diabetes mellitus, which is one of the most prevalent chronic disorders in the modern world. Diabetic retinopathy is one of the secondary complications encountered by the patients suffering from chronic diabetes mellitus. Two major characterizing features of diabetic retinopathy are - macular edema and angiogenesis. It has been noted in the past few years that by controlling or completely inhibiting the factors contributing to the progression of events leading to angiogenesis, there is a noticeable amount of progress seen in the prevention and cure of the animal models of diabetic retinopathy. Endostatin is one such antiangiogenic agent being studied at present. It is a carbon terminal protein fragment obtained after cleavage from the carbon terminus of collagen XVIII. It is one of the most potent inhibitors of angiogenesis known at present and is currently undergoing clinical trials. Although the exact mechanism of action of endostatin is not completely known, various factors which are altered/influenced by the action of endostatin are being studied. These include the downregulation and activation/inactivation of various factors which have been proven to have some role in the progression of angiogenesis. Endostatin could be well exploited as a durable agent in the antiangiogenic therapy, once the clinical trials show positive results.

Compartment-specific expression of collagens and their processing enzymes in intrapulmonary arteries of IPAH patients.

Alterations in extracellular matrix (ECM) have been implicated in the pathophysiology of pulmonary hypertension. Here, we have undertaken a compartment-specific study to elucidate the expression profile of collagens and their processing enzymes in donor and idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries. Predominant intimal, but also medial and perivascular, remodeling and reduced lumen diameter were detected in IPAH pulmonary arteries. Two-photon microscopy demonstrated accumulation of collagen fibers. Quantification of collagen in pulmonary arteries revealed collagen accumulation mainly in the intima of IPAH pulmonary arteries compared with donors. Laser capture-microdissected pulmonary artery profiles (intima+media and perivascular tissue) were analyzed by real-time PCR for ECM gene expression. In the intima+media of IPAH vessels, collagens (COL4A5, COL14A1, and COL18A1), matrix metalloproteinase (MMP) 19, and a disintegrin and metalloprotease (ADAM) 33 were higher expressed, whereas MMP10, ADAM17, TIMP1, and TIMP3 were less abundant. Localization of COLXVIII, its cleavage product endostatin, and MMP10, ADAM33, and TIMP1 was confirmed in pulmonary arteries by immunohistochemistry. ELISA for collagen XVIII/endostatin demonstrated significantly elevated plasma levels in IPAH patients compared with donors, whereas circulating MMP10, ADAM33, and TIMP1 levels were similar between the two groups. Endostatin levels were correlated with pulmonary arterial wedge pressure, and established prognostic markers of IPAH, right atrial pressure, cardiac index, 6-min walking distance, NH2-terminal pro-brain natriuretic peptide, and uric acid. Expression of unstudied collagens, MMPs, ADAMs, and TIMPs were found to be significantly altered in IPAH intima+media. Elevated levels of circulating collagen XVIII/endostatin are associated with markers of a poor prognosis.

Brain malformations associated with Knobloch syndrome--review of literature, expanding clinical spectrum, and identification of novel mutations.

BACKGROUND: Knobloch syndrome is a rare, autosomal recessive, developmental disorder characterized by stereotyped ocular abnormalities with or without occipital skull deformities (encephalocele, bone defects, and cutis aplasia). Although there is clear heterogeneity in clinical presentation, central nervous system malformations, aside from the characteristic encephalocele, have not typically been considered a component of the disease phenotype. METHODS: Four patients originally presented for genetic evaluation of symptomatic structural brain malformations. Whole-genome genotyping, whole-exome sequencing, and confirmatory Sanger sequencing were performed. Using immunohistochemical analysis, we investigated the protein expression pattern of COL18A1 in the mid-fetal and adult human cerebral cortex and then analyzed the spatial and temporal changes in the expression pattern of COL18A1 during human cortical development using the Human Brain Transcriptome database. RESULTS: We identified two novel homozygous deleterious frame-shift mutations in the COL18A1 gene. On further investigation of these patients and their families, we found that many exhibited certain characteristics of Knobloch syndrome, including pronounced ocular defects. Our data strongly support an important role for COL18A1 in brain development, and this report contributes to an enhanced characterization of the brain malformations that can result from deficiencies of collagen XVIII. CONCLUSIONS: This case series highlights the diagnostic power and clinical utility of whole-exome sequencing technology-allowing clinicians and physician scientists to better understand the pathophysiology and presentations of rare diseases. We suggest that patients who are clinically diagnosed with Knobloch syndrome and/or found to have COL18A1 mutations via genetic screening should be investigated for potential structural brain abnormalities even in the absence of an encephalocele.

Collagen XVIII short isoform is critical for retinal vascularization, and overexpression of the Tsp-1 domain affects eye growth and cataract formation.

PURPOSE: Collagen XVIII deficiency leads to anterior and posterior eye defects in Col18a1(-/-) mice, and overexpression of its C-terminal endostatin domain under a K14 promoter leads to cataract. We studied the consequences of K14-driven overexpression of the thrombospondin-1 (Tsp-1)-like domain, and also the roles of the three collagen XVIII isoforms in mice specifically lacking either the promoter 1-derived short or the promoter 2-derived medium/long isoforms. METHODS: Two transgenic lines were generated and compared to Col18a1(-/-) and promoter 1 and 2 knockouts. Enucleated eyes were analyzed histopathologically, immunohistochemically, biochemically, and ultrastructurally. IOP was measured by noninvasive tonometry, and the anterior chamber was studied in vivo using a slit-lamp and optical coherence tomography. RESULTS: Overexpression of the Tsp-1 transgene in an FVB/N background resulted in increased axial length, and substantial incidences of cataract, lens subluxation, phthisis, retinal ablation, corneal vascularization, and intraocular hemorrhages. The FVB/N Col18a1(-/-) mice were affected similarly. The findings in the knockout and transgenic lines were milder in a C57BL/6JOlaHsd (B6) background. Studies with the promoter-specific knockouts revealed the short isoform as the sole variant in the lens capsule and inner limiting membrane, while the ciliary body, iris, and Bruch's membrane contained short and medium/long isoforms. Lack of the short isoform, but not of the medium/long isoforms, caused aberrant retinal vascularization. CONCLUSIONS: An excess of the collagen XVIII Tsp-1 domain is deleterious in the eye, possibly by impairing certain functions of the full-length molecule. Moreover, the short isoform is the critical variant in the development of the posterior eye structures.

Improved stability of multivalent antibodies containing the human collagen XV trimerization domain.

We recently described the in vitro and in vivo properties of an engineered homotrimeric antibody made by fusing the N-terminal trimerization region of collagen XVIII NC1 domain to the C-terminus of a scFv fragment [trimerbody (scFv-NC1) 3; 110 kDa]. Here, we demonstrated the utility of the N-terminal trimerization region of collagen XV NC1 domain in the engineering of trivalent antibodies. We constructed several scFv-based trimerbodies containing the human type XV trimerization domain and demonstrated that all the purified trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Importantly, type XV trimerbodies demonstrated substantially greater thermal and serum stability and resistance to protease digestion than type XVIII trimerbodies. In summary, the small size, high expression level, solubility and stability of the trimerization domain of type XV collagen make it the ideal choice for engineering homotrimeric antibodies for cancer detection and therapy.