Fibrillar Collagen Research Articles

PSIV-6 Differential gene expression of fibro/adipogenic progenitors between Wagyu and Brahman cattle: A possible contribution to their different meat quality

Abstract The amount of intramuscular fat directly influences the meat quality. However, significant differences in the ability to accumulate intramuscular fat are present among different beef cattle breeds. While Wagyu, a cattle breed that originated from Japan, is renowned for abundant intramuscular fat, Brahman cattle generally have very little intramuscular fat accumulation and produce tougher meat. We identified that bovine intramuscular fat is derived from a group of bipotent progenitor cells named fibro/adipogenic progenitors (FAPs) which also give rise to fibroblasts. Thus, the variation in intramuscular fat development between Wagyu and Brahman is likely attributed to the difference in FAPs between these two breeds. In order to understand the gene expression difference between FAPs of the two breeds, single-cell RNA-seq was performed using total single-nucleated cells isolated from the longissimus muscle of young purebred Wagyu, purebred Brahman, and Wagyu-Brahman cross cattle. FAPs constitute the largest single-nucleated cell population in both Wagyu and Brahman skeletal muscle. Multiple subpopulations of FAPs with different gene expression profiles were identified, suggesting that FAP is a heterogeneous population. A unique FAP cluster expressing lower levels of fibrillar collagen and extracellular remodeling enzyme genes but higher levels of select proadipogenic genes was identified exclusively in Wagyu skeletal muscle, which likely contributes to the robust intramuscular adipogenic efficiency of Wagyu FAPs. In conclusion, the difference in the cellular composition and gene expression of FAPs between Wagyu and Brahman cattle likely contribute to their distinct meat quality.

Tyrosine kinase-independent actions of DDR2 in tumor cells and cancer-associated fibroblasts influence tumor invasion, migration and metastasis.

ABSTRACTBoth tumor cell-intrinsic signals and tumor cell-extrinsic signals from cells within the tumor microenvironment influence tumor cell dissemination and metastasis. The fibrillar collagen receptor tyrosine kinase (RTK) discoidin domain receptor 2 (DDR2) is essential for breast cancer metastasis in mouse models, and high expression of DDR2 in tumor and tumor stromal cells is strongly associated with poorer clinical outcomes. DDR2 tyrosine kinase activity has been hypothesized to be required for the metastatic activity of DDR2; however, inhibition of DDR2 tyrosine kinase activity, along with that of other RTKs, has failed to provide clinically relevant responses in metastatic patients. Here, we show that tyrosine kinase activity-independent action of DDR2 in tumor cells can support Matrigel invasion and in vivo metastasis. Paracrine actions of DDR2 in tumor cells and cancer-associated fibroblasts (CAFs) also support tumor invasion, migration and lung colonization in vivo. These data suggest that tyrosine kinase-independent functions of DDR2 could explain failures of tyrosine kinase inhibitor treatment in metastatic breast cancer patients and highlight the need for alternative therapeutic strategies that inhibit both tyrosine kinase-dependent and -independent actions of RTKs in the treatment of breast cancer.This article has an associated First Person interview with the first author of the paper.

Fabrication and characterization methods for investigating cell-matrix interactions in environments possessing spatial orientation heterogeneity

Fibrillar collagen is a ubiquitous structural protein that plays a significant role in determining the mechanical properties of various tissues. The constituent collagen architecture can give direct insight into the respective functional role of the tissue due to the strong structure-function relationship that is exhibited. In such tissues, matrix structure can vary across local subregions contributing to mechanical heterogeneity which can be implicated in tissue function or failure. The post-myocardial infarction scar environment is an example of note where mechanically insufficient collagen can result in impaired cardiac function and possibly tissue rupture due to post-MI cellular response and matrix interactions. In order to further develop the understanding of cell-matrix interactions within heterogeneous environments, we developed a method of heterogeneous collagen gel fabrication which produces a region of randomly oriented fibers directly adjacent to an interconnected region of anisotropic alignment. To fully capture and evaluate the degree of alignment and spatial orientation heterogeneity, several image processing and automated analysis methods were employed. Our analysis revealed the successful fabrication of an interconnected spatially heterogeneous collagen gel possessing distinct regions of random or preferential alignment. Additionally, embedded cell populations were observed to recognize and reorient with their underlying and surrounding architectures through our cell-centric analysis techniques. Statement of significanceFibrillar collagen is a structural protein that contributes to the architecture-function relationship exhibited by various tissues where mechanically insufficient collagen architecture can lead to tissue failure. One environment where this can occur is the post-myocardial infarction scar environment where too much or too little collagen accumulation coupled with spatial fiber orientation heterogeneity can lead to environments incapable of normal mechanical functionality. While there are methodologies capable of generating aligned constructs, they do so with varying degrees of control and complexity with many producing uniform construct alignment. The presented platform is simple and produces continuous constructs possessing inherent spatial orientation heterogeneity. Coupling this with image processing and automated analysis methods enables the probing of fundamental cell-matrix interactions within heterogeneous environments.

Influence of solar radiation insolation precipitation on hydrophobized leather for shoe uppers

The influence of solar radiation and precipitation on leather samples for the upper part of footwear, treated with water-repellent compounds, was determined and the process of their destruction in natural atmospheric conditions was determined. The study of the effect of solar radiation on natural skin has not been sufficiently studied. In connection with the above, the test was carried out on experimental hydrophobized and initial (control) leather samples in an open area to determine the insolation of solar radiation and precipitation on the skin for the upper shoes. During the tests, a systematic examination of the exposed samples was carried out for 1-6 months, sampling for physico-mechanical and other tests was recorded by meteorological conditions. After that, the samples were subjected to organoleptic evaluation, physical-mechanical and chemical-analytical analyses, as well as structural studies. It was visually determined that, depending on the variant of hydrophobization used, fibrillar collagen formations change in different ways. After atmospheric insolation acts on the control sample, its fibrillar structure changes sharply, numerous micro cracks appear, located mainly perpendicular to the fiber axis, and clearly defined dark zones are found between the fibrils. Fibril outlines become indistinct, numerous torn ends are observed. However, such changes are not observed for hydrophobized samples. Significant changes in physical and mechanical properties are observed in the first and fourth months of natural aging. In the hydrophobized sample, the rate of change in hydrothermal destruction and the appearance of cracks in the face layer subjected to aging in an open area, is always lower compared to the control sample.

Real-time polarization microscopy of fibrillar collagen in histopathology

Over the past two decades, fibrillar collagen reorganization parameters such as the amount of collagen deposition, fiber angle and alignment have been widely explored in numerous studies. These parameters are now widely accepted as stromal biomarkers and linked to disease progression and survival time in several cancer types. Despite all these advances, there has not been a significant effort to make it possible for clinicians to explore these biomarkers without adding steps to the clinical workflow or by requiring high-cost imaging systems. In this paper, we evaluate previously described polychromatic polarization microscope (PPM) to visualize collagen fibers with an optically generated color representation of fiber orientation and alignment when inspecting the sample by a regular microscope with minor modifications. This system does not require stained slides, but is compatible with histological stains such as H&E. Consequently, it can be easily accommodated as part of regular pathology review of tissue slides, while providing clinically useful insight into stromal composition.

Crtap and p3h1 knock out zebrafish support defective collagen chaperoning as the cause of their osteogenesis imperfecta phenotype

Prolyl 3-hydroxylation is a rare collagen type I post translational modification in fibrillar collagens. The primary 3Hyp substrate sites in type I collagen are targeted by an endoplasmic reticulum (ER) complex composed by cartilage associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and prolyl cis/trans isomerase B, whose mutations cause recessive forms of osteogenesis imperfecta with impaired levels of α1(I)3Hyp986. The absence of collagen type I 3Hyp in wild type zebrafish provides the unique opportunity to clarify the role of the complex in vertebrate. Zebrafish knock outs for crtap and p3h1 were generated by CRISPR/Cas9. Mutant fish have the typical OI patients' reduced size, body disproportion and altered mineralization. Vertebral body fusions, deformities and fractures are accompanied to reduced size, thickness and bone volume. Intracellularly, collagen type I is overmodified, and partially retained causing enlarged ER cisternae. In the extracellular matrix the abnormal collagen type I assembles in disorganized fibers characterized by altered diameter. The data support the defective chaperone role of the 3-hydroxylation complex as the primary cause of the skeletal phenotype.

Design and Production of Customizable and Highly Aligned Fibrillar Collagen Scaffolds.

The ability to fabricate anisotropic collagenous materials rapidly and reproducibly has remained elusive despite decades of research. Balancing the natural propensity of monomeric collagen (COL) to spontaneously polymerize in vitro with the mild processing conditions needed to maintain its native substructure upon polymerization introduces challenges that are not easily amenable with off-the-shelf instrumentation. To overcome these challenges, we have designed a platform that simultaneously aligns type I COL fibrils under mild shear flow and builds up the material through layer-by-layer assembly. We explored the mechanisms propagating fibril alignment, targeting experimental variables such as shear rate, viscosity, and time. Coarse-grained molecular dynamics simulations were also employed to help understand how initial reaction conditions including chain length, indicative of initial polymerization, and chain density, indicative of concentration, in the reaction environment impact fibril growth and alignment. When taken together, the mechanistic insights gleaned from these studies inspired the design, iteration, fabrication, and then customization of the fibrous collagenous materials, illustrating a platform material that can be readily adapted to future tissue engineering applications.

Гліциризинова кислота: патофізіологічні аспекти формування фіброзу та ефективність у лікуванні захворювань печінки

В оглядовій статті наведені сучасні дані щодо чинників виникнення та розвитку фіброзу печінки. Цей стан обумовлений надмірним накопиченням позаклітинного матриксу, продуцентом якого є здебільшого печінкові зірчасті клітини, які при активації набувають можливості продукувати патологічний колаген І та ІІІ типів. Відкладання фібрилярної тканини та колагену у просторі Діссе призводить до появи базальної мембрани в епітелії синусоїдів, унаслідок чого відбувається їх капіляризація. Портальна гіпертензія поглиблюється виникненням ендотеліальної дисфункції та патологічною продукцією вазоконстрикторів. Подальше прогресування фіброзу печінки є фактором ризику виникнення цирозу та навіть гепатоцелюлярної карциноми. Наведені дані щодо досліджень антифібротичних властивостей гліциризину — препарату рослинного походження. Майже 40-річна історія офіційного вивчення довела його протизапальні, противірусні, антифібротичні властивості. Дія гліциризину відбувається завдяки впливу на імунні клітини, гальмуванню апоптозу гепатоцитів та прискоренню апоптозу патологічно активованих зірчастих клітин. Вибірковою дією на фактори транскрипції можна пояснити протиканцерогенний ефект препарату при тривалому перебігу вірусних гепатитів.

In utero exposure to electronic-cigarette aerosols decreases lung fibrillar collagen content, increases Newtonian resistance and induces sex-specific molecular signatures in neonatal mice

Approximately 7% of pregnant women in the United States use electronic-cigarette (e-cig) devices during pregnancy. There is, however, no scientific evidence to support e-cig use as being ‘safe’ during pregnancy. Little is known about the effects of fetal exposures to e-cig aerosols on lung alveologenesis. In the present study, we tested the hypothesis that in utero exposure to e-cig aerosol impairs lung alveologenesis and pulmonary function in neonates. Pregnant BALB/c mice were exposed 2 h a day for 20 consecutive days during gestation to either filtered air or cinnamon-flavored e-cig aerosol (36 mg/mL of nicotine). Lung tissue was collected in offspring during lung alveologenesis on postnatal day (PND) 5 and PND11. Lung function was measured at PND11. Exposure to e-cig aerosol in utero led to a significant decrease in body weights at birth which was sustained through PND5. At PND5, in utero e-cig exposures dysregulated genes related to Wnt signaling and epigenetic modifications in both females (~ 120 genes) and males (40 genes). These alterations were accompanied by reduced lung fibrillar collagen content at PND5—a time point when collagen content is close to its peak to support alveoli formation. In utero exposure to e-cig aerosol also increased the Newtonian resistance of offspring at PND11, suggesting a narrowing of the conducting airways. At PND11, in females, transcriptomic dysregulation associated with epigenetic alterations was sustained (17 genes), while WNT signaling dysregulation was largely resolved (10 genes). In males, at PND11, the expression of only 4 genes associated with epigenetics was dysregulated, while 16 Wnt related-genes were altered. These data demonstrate that in utero exposures to cinnamon-flavored e-cig aerosols alter lung structure and function and induce sex-specific molecular signatures during lung alveologenesis in neonatal mice. This may reflect epigenetic programming affecting lung disease development later in life.

Fibrillar Collagen: A Review of the Mechanical Modeling of Strain-Mediated Enzymatic Turnover

AbstractCollagen is the most abundant protein in mammals, often serving as the main load bearing constituent in soft tissues. Collagen undergoes continuous remodeling processes in which present collagen degrades, and new collagen is formed and deposited. Experiments find that modestly strained fibrillar collagen is often stabilized to enzymatic degradation, a mechanism that is essential in approaching and maintaining a homeostatic balance in the tissue remodeling process for healthy tissue. At larger strains, this decline in the collagen degradation rate may be reversed. This article reviews different modeling approaches that seek to account for the effect of straining on collagen remodeling, both with respect to collagen amount and to resulting mechanical properties. These models differ in the considered length scale starting from the molecular scale up to the larger continuum scale.

Abstract P104: Simultaneous Treatment With Ivabradine And Spironolactone Does Not Modify Hypertension-induced Myocardial Fibrosis In Dahl Salt-sensitive Rats

Background: Hypertension (HT)-induced accumulation of myocardial collagen stiffens the left ventricular (LV) wall and, hence, promotes diastolic dysfunction. Considering that a competitive aldosterone receptor antagonist spironolactone (SL) as well as a “pure” heart rate-lowering drug ivabradine (IVA) have both been proven to exert an anti-fibrotic effect during cardiac remodeling, we hypothesized that, in combination, these drugs would complement each other in alleviating HT-induced myocardial fibrosis and, therefore, could better preserve diastolic function. Methods: Sustained HT was induced in 7-week-old male Dahl salt-sensitive rats by feeding them a high-salt (8%) diet for 7 weeks. Then, HT rats were randomly assigned in two experimental groups: 1) IVA (10 mg/kg/day) + SL (20 mg/kg/day)-treated (HT-T) and 2) vehicle only (HT-V). The drugs and the vehicle (a mixture of 50% dimethyl sulfoxide/50% propylene glycol, v/v) were delivered IP by osmotic pumps for 8 weeks. The age-matched rats were used as normotensive controls (NT). Heart rate (HR) and blood pressure (BP) were recorded every other week using a CODA tail-cuff plethysmography system. At the end of the study, the hemodynamic parameters were assessed using a Millar pressure catheter and the hearts were collected for histology and quantitative morphometry. Statistical analysis was performed using Prism 6. Results: We found that during 8-week treatment period, BP had remained comparably elevated in both HT groups by ~26% (P≤0.01) on average vs. NT rats, whereas HR had been persistently reduced in HT-T group by ~38% and ~27% (P≤0.01) on average vs. HT-V and NT rats, respectively. At the end of the study period, all groups revealed a similar level of LV end-diastolic pressure, suggesting the analogous diastolic stiffness of the myocardium. At the same time, morphological examination of the hearts has confirmed that sustained HT caused significant interstitial and perivascular fibrosis (P≤0.01) in both experimental groups compared to NT rats, and that the treatment did not change the extent of fibrillar collagen accumulation as compared to HT-V rats. Conclusions: Taken together, our findings confirmed that a combined treatment with IVA and SL could not alleviate HT-induced myocardial fibrosis.

Heterogeneity in proline hydroxylation of fibrillar collagens observed by mass spectrometry.

Collagen is the major protein in the extracellular matrix and plays vital roles in tissue development and function. Collagen is also one of the most processed proteins in its biosynthesis. The most prominent post-translational modification (PTM) of collagen is the hydroxylation of Pro residues in the Y-position of the characteristic (Gly-Xaa-Yaa) repeating amino acid sequence of a collagen triple helix. Recent studies using mass spectrometry (MS) and tandem MS sequencing (MS/MS) have revealed unexpected hydroxylation of Pro residues in the X-positions (X-Hyp). The newly identified X-Hyp residues appear to be highly heterogeneous in location and percent occupancy. In order to understand the dynamic nature of the new X-Hyps and their potential impact on applications of MS and MS/MS for collagen research, we sampled four different collagen samples using standard MS and MS/MS techniques. We found considerable variations in the degree of PTMs of the same collagen from different organisms and/or tissues. The rat tail tendon type I collagen is particularly variable in terms of both over-hydroxylation of Pro in the X-position and under-hydroxylation of Pro in the Y-position. In contrast, only a few unexpected PTMs in collagens type I and type III from human placenta were observed. Some observations are not reproducible between different sequencing efforts of the same sample, presumably due to a low population and/or the unpredictable nature of the ionization process. Additionally, despite the heterogeneous preparation and sourcing, collagen samples from commercial sources do not show elevated variations in PTMs compared to samples prepared from a single tissue and/or organism. These findings will contribute to the growing body of information regarding the PTMs of collagen by MS technology, and culminate to a more comprehensive understanding of the extent and the functional roles of the PTMs of collagen.

Progressive alcohol-related liver fibrosis is characterised by imbalanced collagen formation and degradation.

SummaryBackgroundLiver fibrosis accumulation is considered a turnover disease, with formation exceeding degradation, although this hypothesis has never been tested in humans.AimsTo investigate extracellular matrix (ECM) remodelling in a biopsy‐controlled study of alcohol‐related liver disease (ALD) patients.MethodsWe evaluated the relationship between formation and degradation of four collagens as a function of histological fibrosis, inflammation and steatosis in 281 patients with ALD and 50 matched healthy controls. Post hoc, we tested the findings in a cohort of patients with alcohol‐related cirrhosis and assessed the collagens' prognostic accuracy. We assessed the fibrillar collagens type III (PRO‐C3/C3M) and V (PRO‐C5/C5M), the basement membrane collagen IV (PRO‐C4/C4M), and the microfilament interface collagen VI (PRO‐C6/C6M).ResultsMean age was 54 ± 6 years, 74% male, fibrosis stage F0/1/2/3/4 = 33/98/84/18/48. Compared to controls, patients with ALD had higher levels of type III collagen formation and degradation, with the highest concentrations in those with cirrhosis (PRO‐C3 = 8.2 ± 1.7 ng/mL in controls, 14.6 ± 13.5 in ALD, 34.8 ± 23.1 in cirrhosis; C3M 7.4 ± 1.9 in controls, 9.3 ± 4.4 in ALD, 14.0 ± 5 in cirrhosis). ECM remodelling became increasingly imbalanced in higher stages of liver fibrosis, with formation progressively superseding degradation. This was particularly pronounced for type III collagen. We observed similar imbalance for inflammatory severity, but not steatosis.ConclusionsALD is characterised by both elevated collagen formation and degradation, which becomes increasingly imbalanced with more severe disease. Net increase in fibrillar collagens contributes to fibrosis progression. This has important implications for monitoring and very early identification of patients at highest risk of progressing to cirrhosis.

Genipin Delays Corneal Stromal Enzymatic Digestion.

PurposeTo evaluate the use of genipin in delaying enzymatic digestion of corneal stroma.MethodsHuman corneal stromal tissue was treated with genipin, a known chemical crosslinker, and then along with control tissue was subjected to enzymatic digestion with collagenase. The effects of genipin treatment in retarding stromal digestion were analyzed with phase contrast microscopy, a protein quantification assay, second harmonic generation imaging, and transmission electron microscopy.ResultsGenipin increased stromal resistance to enzymatic digestion when compared with untreated stroma. A morphologic analysis and protein quantification showed increased stromal resistance to enzymatic digestion once stromal tissue was treated with genipin. Second harmonic generation imaging revealed persistent fibrillar collagen signaling in genipin-treated tissue in contrast with untreated tissue suggesting that genipin retards collagenolysis.ConclusionsGenipin increases stromal resistance to enzymatic digestion in controlled experiments as demonstrated by protein quantification studies and through morphologic imaging.Translational RelevanceThis study explores the novel use of genipin in delaying enzymatic stromal digestion. Delaying stromal melting in the setting of corneal infectious or autoimmune keratitis can potentially decrease clinical morbidity.

P1. Efficacy of a calcium phosphate collagen matrix bone graft with needle-shaped submicron surface topography in a rabbit posterolateral lumbar spine fusion model

<h3>BACKGROUND CONTEXT</h3> Synthetic bone grafts are a promising alternative for the treatment of degenerative spine conditions and are available without the added drawbacks associated autografts including limited availability and donor-site morbidity. Of these synthetic materials, calcium phosphate (CaP) with a characteristic needle-shaped submicron surface topography (BCP<µm) has attracted much attention due to its unique bone-forming ability which is essential for repairing critical-size bone defects such as those found in the posterolateral spine [1,2]. Previous in vitro and ex-vivo data presented by van Dijk LA [3] and Yuan H [4] at NASS, 2020 demonstrated that these specific surface characteristics drive a favorable response from the innate immune system. <h3>PURPOSE</h3> This study was to evaluate the efficacy of a novel synthetic bone graft using a clinically-relevant "Boden" rabbit posterolateral spine fusion model. The synthetic bone graft comprised of CaP granules with a needle-shaped submicron surface structure (BCP<µm) embedded in a highly porous and fibrillar bovine type I collagen matrix (col-BCP<µm). The fibrillar collagen was introduced as a carrier, designed to facilitate admixing of the graft with biological fluids perioperatively and to minimize graft migration postoperatively. <h3>STUDY DESIGN/SETTING</h3> The novel synthetic bone graft was first mixed in a 1:1 volume ratio with autologous bone (ABG). Subsequently, grafts were implanted in the rabbit posterolateral spine environment for 3 days, 6, 9 and 12 weeks. An autologous bone graft group was used as a positive control in this study. Outcomes for the test article were additionally compared to historical data for the same material (BCP<µm) implanted alone, without the fibrillar collagen carrier. <h3>OUTCOME MEASURES</h3> Spinal fusion was determined by manual palpation (MP), X-ray and µCT imaging (based on the Lenke scale), mechanical testing (range of motion testing at 12 weeks only) and histology (decalcified and undecalcified). <h3>METHODS</h3> Forty skeletally mature New Zealand white rabbits were divided into two groups and underwent single-level bilateral posterolateral intertransverse process spine arthrodesis at L4-5. Animals were euthanized after 3 days, 6, 9 and 12 weeks. <h3>RESULTS</h3> Based on all endpoints, the test group and positive control group showed an equivalent and gradual progression in bone formation and implant resorption over time, leading to mature fusion masses at 12 weeks. No adverse reactions were observed in any of the groups. Bilateral fusion rates by MP were consistent with the literature (0-40% at 6 weeks; 60-70% at 12 weeks) and similar trends were seen by histological and radiographic assessment (both X-ray and µCT). Flexion-extension and lateral bending results at 12 weeks demonstrated fusion of the treated level consistent with the MP results. The results observed for the test group were consistent with those seen historically for the same material implanted without the fibrillar collagen carrier. <h3>CONCLUSIONS</h3> These results show that BCP with needle-shaped submicron-scale surface topography (BCP<µm) combined with a fibrillar collagen matrix (col-BCP<µm) could achieve equivalent fusion rates to ABG when used as a bone graft extender in a clinically relevant single level rabbit intertransverse process posterolateral lumbar fusion model. Over time, a progression in bone healing, graft resorption and remodeling were seen with all experimental endpoints, with equivalency between the groups. The results for col-BCP<µm were consistent with those previously seen for BCP<µm alone, confirming that the fibrillar collagen carrier improved handling properties but was not detrimental to the bone-forming capacity of the BCP<µm. <h3>FDA DEVICE/DRUG STATUS</h3> Col-BCP<µm (Investigational/Not approved)

In silico trials to design potent inhibitors against matrilysin (MMP-7)

The study deals with structure-based rational drug design against the chief zinc-rely endopeptidase called matrilysin (MMP-7) that is involved in inflammatory and metastasis process of several carcinomas. Hyperactivated matrilysin of human was targeted, because of its hydrolytic actions on extracellular matrix (ECM) protein components constitutes fibrillar collagens, gelatins, fibronectins and it also activates zymogen forms of vital matrix metalloproteinases (gelatinase A-MMP-2 and B-MMP-9) responsible for ECM destruction in many cancers. In the present work, e-pharmacophores were generated for the respective five co-crystal structures of human matrilysin by mapping ligand’s pharmacophoric features. During the lead-optimization campaign, the five e-pharmacophores-based shape screening against an in-house library of >21 million compounds created a dataset of 5000 structural analogs. The subsequent three different docking strategies, including rigid-receptor docking, quantum-polarized-ligand docking, induced-fit docking and free energy binding calculations resulted four leads as novel and potent MMP-7 binders. These four leads were observed with good pharmacological features and good receiver operating characteristics curve metrics (ROC: 0.93) in post-docking evaluations against five existing co-crystal inhibitors and 1000 decoy molecules with MMP-7. Moreover, stability and dynamics behavior of matrilysin-lead1 complex and matrilysin-cocrystal ligand (TQJ) complex were analyzed in natural physiological milieu of 1000 ns or 1 µs molecular dynamics simulations. Lead1-MMP-7 complex was found with an average Cα root-mean-square deviation (RMSD) of 2.35 Å, average ligand root-mean-square fluctuations (RMSF) of 0.66 Å and the strong metallic interactions with E220, a key residue for proteolytic action thereby hinders ECM proteolysis that in turn can halt metastatic cancerous condition. Communicated by Ramaswamy H. Sarma

Deficiency of Procollagen C-Proteinase Enhancer 1 in Mice has No Major Impact on Cardiac Collagen and Function Under Basal Conditions.

Maturation of fibrillar collagen is known to play a crucial role in the pathophysiology of myocardial fibrosis. Procollagen C-proteinase enhancer 1 (PCPE1) has a key role in procollagen maturation and collagen fibril formation. The phenotype of both male and female PCPE1 knock-out mice was investigated under basal conditions to explore the potential of PCPE1 as a therapeutic target in heart failure. Global constitutive PCPE1-/- mice were generated. Serum procollagen I C-terminal propeptide, organ histology, and cutaneous wound healing were assessed in both wild type (WT) and PCPE1-/- mice. In addition, the cardiac expression of genes involved in collagen metabolism was investigated and the total and insoluble cardiac collagen contents determined. Cardiac function was evaluated by echocardiography. No differences in survival, clinical chemistry, or organ histology were observed in PCPE1-/- mice compared with WT. Serum procollagen I C-terminal propeptide was lower in PCPE1-/- mice. Cardiac mRNA expression of Bmp1, Col1a1, Col3a1, and Loxl2 was similar, whereas Tgfb and Loxl1 mRNA levels were decreased in PCPE1-/- mice compared with sex-matched WT. No modification of total or insoluble cardiac collagen content was observed between the 2 strains. Ejection fraction was slightly decreased in PCPE1-/- male mice, but not in females. Finally, wound healing was not altered in PCPE1-/- mice. PCPE1 deficiency does not trigger any major liabilities and does not affect cardiac collagen content nor its function under basal conditions. Further studies are required to evaluate its role under stressed conditions and determine its suitability as a therapeutic target for heart failure.

Preventing ovarian cancer cell metastasis through inhibition of discoidin domain receptor 2 (DDR2) in the tumor microenvironment: a metabolomic analysis

Objectives: Discoidin domain receptor 2 (DDR2) is a tyrosine kinase activated by fibrillar collagen type 1. DDR2 expression in both cancer cells and stromal cells is critical for metastases in breast cancer. In ovarian cancer, DDR2 stabilizes Snail, a critical factor in the epithelial to mesenchymal transition, which facilitates cancer cell invasion and metastases. Snail has also been shown to reprogram glucose metabolism in breast cancer cells, thereby allowing cancer cell survival under metabolic stress. This study aimed to investigate how stromal DDR2 expression in normal omental and ovarian cancer associated fibroblasts (NOF and CAF) regulates tumor cell invasion by analyzing changes in stromal cell metabolomics. Methods: Matrigel invasion assays were performed using immortalized normal omental fibroblasts (NOF206) and ES2 ovarian cancer cells. NOF206s were transfected with two different siRNAs to produce transient siDDR2 knockdown. Lysates and conditioned media were collected of siControl (DDR2-expressing), Dharmacon siDDR2 and Sigma siDDR2 (DDR2 non-expressing) cells. Mass spectrometry was performed and metabolites were identified. Quantitative RT-PCR was performed to evaluate changes in the expression of mesenchymal markers based on DDR2 expression. Results: DDR2 expressing tumor cells invaded less when co-cultured with DDR2-non expressing NOFs compared to DDR2 expressing NOFs (51 vs 155 cells per hpf, p Conclusions: Genetic inactivation of DDR2 results in marked changes in the metabolism of normal omental fibroblasts resulting in decreased tumor cell invasion. These findings suggest a novel role of DDR2 in metabolism and stromal cell energy generation. The significance of these findings requires further study on the complex relationship between ovarian cancer and the stromal environment.

Discoidin Domain Receptor 2 (DDR2) expression in combination with mutation status is predictive of survival in patients with ovarian cancer

<h3>Objectives:</h3> Dicoidin Domain Receptor 2 (DDR2) is a tyrosine kinase receptor which binds the most common extracellular matrix protein, fibrillar collagen type 1. Expression of DDR2 is critical for invasion and migration of ovarian cancer tumor cells. Clinically, high DDR2 expression has been shown to be associated with advanced stage disease and decreased survival. It is not known how <i>BRCA</i> mutation status, a known marker for treatment sensitivity, impacts this relationship between DDR2 expression and survival. This study aimed to determine whether DDR2 expression was correlated with <i>BRCA</i> mutation status and to quantify the effects of this relationship on survival. <h3>Methods:</h3> Immunohistochemical (IHC) analysis of DDR2 expression in two ovarian cancer tissue microarrays (TMA) was performed. DDR2 staining on the membrane of tumor cells was scored microscopically according to the intensity and percentage of cells positive for DDR2 expression compared to controls. High DDR2 staining was defined as >70% and low staining <69%. Clinical information, including germline mutational status and survival data, was abstracted from the electronic medical record. Statistical analysis was performed using descriptive statistics and unpaired t-tests. Survival analysis was performed using the Kaplan-Meier method with statistical significance determined using the log-rank test. <h3>Results:</h3> The cohort included 164 patients diagnosed between 2/1994 and 1/2017. The majority had advanced stage disease (79%) and high-grade serous histology (76%). Germline mutation status was known in 93 patients (57%). <i>BRCA1</i> was the most common mutation in 38 patients (64%) followed by <i>BRCA2</i> in 20 patients (34%). 34 patients had negative germline testing. DDR2 expression was significantly associated with mutational status. Patients without a germline mutation had significantly higher DDR2 expression (64%) compared to <i>BRCA1</i> (37%) (p=0.002) and <i>BRCA2</i> had significantly higher DDR2 expression (55%) compared to <i>BRCA1</i> (37%) (p=0.03). DDR2 expression did not vary by stage, grade or platinum sensitive status. In survival analysis, patients with high DDR2 expression had significantly lower overall survival (33mo vs 83mo, HR 1.94, p=0.002) and progression free survival (17mo vs 34mo, HR 1.668, p=0.04) compared to patients with low DDR2 expression. When survival analysis was stratified by mutation status and DDR2 expression, survival differences were seen. In patients without a germline mutation, high DDR2 expression was associated with worse overall survival (42mo vs 147mo, HR=2.818, p=0.04) compared to low DDR2 expression. <h3>Conclusions:</h3> Tumor DDR2 expression is correlated with mutation status and when combined, provides useful prognostic information. Studies are ongoing to identify additional <i>BRCA</i> deficient tumors for further survival analysis.

Increasing sensitivity to olaparib through inhibition of discoidin domain receptor 2 (DDR2) in homologous-recombination proficient ovarian cancer models

<h3>Objectives:</h3> Discoidin Domain Receptor 2 (DDR2) is a tyrosine kinase receptor which binds the most common extracellular matrix protein, fibrillar collagen type 1. DDR2 expression is critical for invasion and migration of ovarian cancer tumor cells, making DDR2 a potential target for new treatments. This study aimed to determine whether genetic inactivation of DDR2 in ovarian cancer cells would increase sensitivity to treatment with PARP inhibitor. <h3>Methods:</h3> Two ovarian cancer cells lines, ES2 and COV362, were used for <i>in vitro</i> assays. Stable hairpin control and DDR2 knockdowns (shSCRM and shDDR2) were used for ES2 and transient small interfering RNA knockdown of DDR2 (siControl and siDDR2) were used for COV362. Homologous recombination (HR) status was determined by quantification of RAD-51 foci relative to controls using immunofluorescence after irradiation. Cell viability and survival after treatment with olaparib was quantified using MTS assays and clonogenics. The DNA damage response after treatment with olaparib was analyzed using immunofluorescence. <i>In vivo</i> studies to evaluate the sensitivity to olaparib in the presence or absence of DDR2 expression were performed in nude mice after intraperitoneal injection with ES2shSCRM or shDDR2 cells. <h3>Results:</h3> ES2 and COV362 cells were found to have a 2-fold increase in RAD51 foci after irradiation indicating HR proficiency. DDR2 genetically inactivated cells (ES2shDDR2) were found to be more HR deficient than DDR2 expressing cells (ES2shSCRM) which remained HR proficient (0.80 vs 4.78 relative RAD51 foci per cell). We found that genetic inactivation of DDR2 in the ES2s and COV362s led to increased sensitivity to olaparib with an 80% (15uM vs 73uM) and 68% (62uM vs 195uM) reduction in IC50s, respectively, compared to DDR2 expressing cells. Additionally, ES2shDDR2 cells had decreased cell survival by clonogenic assay after treatment with olaparib compared to ES2shSCRM cells as measured by absorbance (0.64 vs 0.80, p=0.005). We found that DNA damage and the repair response was altered in DDR2 genetically inactivated cells treated with olaparib with an increase in gamma-H2AX (8 vs 6 foci per cell, p<0.0001) and 53BP1 foci (3 vs 1 foci per cell, p<0.0001) when compared to DDR2 expressing cells. In an intraperitoneal metastatic mouse model with the HR-proficient cell line ES2, we found that genetic inactivation of DDR2 led to decreased tumor burden when treated with olaparib compared to DDR2-expressing cells treated with olaparib (1499mm³ vs 3236mm³, p=0.04). <h3>Conclusions:</h3> DDR2 knockdown sensitizes HR proficient ovarian cancer cells to treatment with PARP inhibitor in both <i>in vitro</i> and <i>in vivo</i> models through a mechanism of induced HR deficiency and increased DNA damage. Future experiments will explore whether a DDR2 inhibitor can improve sensitivity to PARP inhibitor in HR proficient ovarian cancer models.