Angiopoietin-like protein 2 regulates endothelial colony forming cell vasculogenesis

Angiopoietin-like 2 (ANGPTL2) has been reported to induce sprouting angiogenesis; however, its role in vasculogenesis, the de novo lumenization of endothelial cells (EC), remains unexplored. We sought to investigate the potential role of ANGPTL2 in regulating human cord blood derived endothelial colony forming cell (ECFC) vasculogenesis through siRNA mediated inhibition of ANGPTL2 gene expression. We found that ECFCs in which ANGPTL2 was diminished displayed a threefold decrease in in vitro lumenal area whereas addition of exogenous ANGPTL2 protein domains to ECFCs lead to increased lumen formation within a 3 dimensional (3D) collagen assay of vasculogenesis. ECFC migration was attenuated by 36 % via ANGPTL2 knockdown (KD) although proliferation and apoptosis were not affected. We subsequently found that c-Jun NH2-terminal kinase (JNK), but not ERK1/2, phosphorylation was decreased upon ANGPTL2 KD, and expression of membrane type 1 matrix metalloproteinase (MT1-MMP), known to be regulated by JNK and a critical regulator of EC migration and 3D lumen formation, was decreased in lumenized structures in vitro derived from ANGPTL2 silenced ECFCs. Treatment of ECFCs in 3D collagen matrices with either a JNK inhibitor or exogenous rhTIMP-3 (an inhibitor of MT1-MMP activity) resulted in a similar phenotype of decreased vascular lumen formation as observed with ANGPTL2 KD, whereas stimulation of JNK activity increased vasculogenesis. Based on gene silencing, pharmacologic, cellular, and biochemical approaches, we conclude that ANGPTL2 positively regulates ECFC vascular lumen formation likely through its effects on migration and in part by activating JNK and increasing MT1-MMP expression

Murine Ultrasound-Guided Transabdominal Para-Aortic Injections of Self-Assembling Type I Collagen Oligomers

Abdominal aortic aneurysms (AAAs) represent a potentially life-threatening condition that predominantly affects the infrarenal aorta. Several preclinical murine models that mimic the human condition have been developed and are now widely used to investigate AAA pathogenesis. Cell- or pharmaceutical-based therapeutics designed to prevent AAA expansion are currently being evaluated with these animal models, but more minimally invasive strategies for delivery could improve their clinical translation. The purpose of this study was to investigate the use of self-assembling type I collagen oligomers as an injectable therapeutic delivery vehicle in mice. Here we show the success and reliability of a para-aortic, ultrasound-guided technique for injecting quickly-polymerizing collagen oligomer solutions into mice to form a collagen-fibril matrix at body temperature. A commonly used infrarenal mouse AAA model was used to determine the target location of these collagen injections. Ultrasound-guided, closed-abdominal injections supported consistent delivery of collagen to the area surrounding the infrarenal abdominal aorta halfway between the right renal artery and aortic trifurcation into the iliac and tail arteries. This minimally invasive approach yielded outcomes similar to open-abdominal injections into the same region. Histological analysis on tissue removed on day 14 post-operatively showed minimal in vivo degradation of the self-assembled fibrillar collagen and the majority of implants experienced minimal inflammation and cell invasion, further confirming this material's potential as a method for delivering therapeutics. Finally, we showed that the typical length and position of this infrarenal AAA model was statistically similar to the length and targeted location of the injected collagen, increasing its feasibility as a localized therapeutic delivery vehicle. Future preclinical and clinical studies are needed to determine if specific therapeutics incorporated into the self-assembling type I collagen matrix described here can be delivered near the aorta and locally limit AAA expansion.

Amine functionalization of cholecyst-derived extracellular matrix with generation 1 PAMAM dendrimer

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Biomacromolecules, copyright © American Chemical Society after peer review. To access the final edited and published work, see http://pubs.acs.org/doi/pdf/10.1021/bm701055k.A method to functionalize cholecyst-derived extracellular matrix (CEM) with free amine groups was established in an attempt to improve its potential for tethering of bioactive molecules. CEM was incorporated with Generation-1 polyamidoamine (G1 PAMAM) dendrimer by using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide cross-linking system. The nature of incorporation of PAMAM dendrimer was evaluated using shrink temperature measurements, Fourier transform infrared (FTIR) assessment, ninhydrin assay, and swellability. The effects of PAMAM incorporation on mechanical and degradation properties of CEM were evaluated using a uniaxial mechanical test and collagenase degradation assay, respectively. Ninhydrin assay and FTIR assessment confirmed the presence of increasing free amine groups with increasing quantity of PAMAM in dendrimer-incorporated CEM (DENCEM) scaffolds. The amount of dendrimer used was found to be critical in controlling scaffold degradation, shrink temperature, and free amine content. Cell culture studies showed that fibroblasts seeded on DENCEM maintained their metabolic activity and ability to proliferate in vitro. In addition, fluorescence cell staining and scanning electron microscopy analysis of cell-seeded DENCEM showed preservation of normal fibroblast morphology and phenotype

Quantitative Analysis of the Effect of Cancer Invasiveness and Collagen Concentration on 3D Matrix Remodeling

Extracellular matrix (ECM) remodeling is a key component of cell migration and tumor metastasis, and has been associated with cancer progression. Despite the importance of matrix remodeling, systematic and quantitative studies on the process have largely been lacking. Furthermore, it remains unclear if the disrupted tensional homeostasis characteristic of malignancy is due to initially altered ECM and tissue properties, or to the alteration of the tissue by tumor cells. To explore these questions, we studied matrix remodeling by two different prostate cancer cell lines in a three-dimensional collagen system. Over one week, we monitored structural changes in gels of varying collagen content using confocal reflection microscopy and quantitative image analysis, tracking metrics of fibril fraction, pore size, and fiber length and diameter. Gels that were seeded with no cells (control), LNCaP cells, and DU-145 cells were quantitatively compared. Gels with higher collagen content initially had smaller pore sizes and higher fibril fractions, as expected. However, over time, LNCaP- and DU-145-populated matrices showed different structural properties compared both to each other and to the control gels, with LNCaP cells appearing to favor microenvironments with lower collagen fiber fractions and larger pores than DU-145 cells. We posit that the DU-145 cells' preference for denser matrices is due to their higher invasiveness and proteolytic capabilities. Inhibition of matrix proteases resulted in reduced fibril fractions for high concentration gels seeded with either cell type, supporting our hypothesis. Our novel quantitative results probe the dynamics of gel remodeling in three dimensions and suggest that prostate cancer cells remodel their ECM in a synergistic manner that is dependent on both initial matrix properties as well as their invasiveness

Marine, Estuarine, and Diadromous Fish Stocks at Risk of Extinction in North America (Exclusive of Pacific Salmonids)

The problem of extinction in the sea has been recognized only recently by the scientific community (Roberts and Hawkins 1999). For the last decade, the American Fisheries Society (AFS) has pursued an initiative to identify marine fish stocks that may be at risk of extinction in North America (Musick 1998; Coleman et al. 2000; Musick et al. 2000a; Musick et al. 2000b; Parker et al. 2000). The present paper presents the first recognized AFS list of marine, estuarine, and diadromous fishes that may be at risk of extinction in North America (exclusive of Pacific salmonids). The geographic scope of this review includes the coastal waters of Mexico, the continental United States, Alaska, and Canada. The objectives in preparing this list have been to identify stocks at risk at a sufficiently early stage of decline to avoid listing as threatened or endangered, minimize the probability of under- or overestimating the risk of extinction, use the best existing knowledge of stock dynamics at low population levels, and allow experts on stocks to bring to bear all the available knowledge about life history and conservation status in order to categorize risk of extinction

Functional tissue engineering of ligament healing

Ligaments and tendons are dense connective tissues that are important in transmitting forces and facilitate joint articulation in the musculoskeletal system. Their injury frequency is high especially for those that are functional important, like the anterior cruciate ligament (ACL) and medial collateral ligament (MCL) of the knee as well as the glenohumeral ligaments and the rotator cuff tendons of the shoulder. Because the healing responses are different in these ligaments and tendons after injury, the consequences and treatments are tissue- and site-specific. In this review, we will elaborate on the injuries of the knee ligaments as well as using functional tissue engineering (FTE) approaches to improve their healing. Specifically, the ACL of knee has limited capability to heal, and results of non-surgical management of its midsubstance rupture have been poor. Consequently, surgical reconstruction of the ACL is regularly performed to gain knee stability. However, the long-term results are not satisfactory besides the numerous complications accompanied with the surgeries. With the rapid development of FTE, there is a renewed interest in revisiting ACL healing. Approaches such as using growth factors, stem cells and scaffolds have been widely investigated. In this article, the biology of normal and healing ligaments is first reviewed, followed by a discussion on the issues related to the treatment of ACL injuries. Afterwards, current promising FTE methods are presented for the treatment of ligament injuries, including the use of growth factors, gene delivery, and cell therapy with a particular emphasis on the use of ECM bioscaffolds. The challenging areas are listed in the future direction that suggests where collection of energy could be placed in order to restore the injured ligaments and tendons structurally and functionally

Murine Ultrasound-Guided Transabdominal Para-Aortic Injections of Self-Assembling Type I Collagen Oligomers

Abdominal aortic aneurysms (AAAs) represent a potentially life-threatening condition that predominantly affects the infrarenal aorta. Several preclinical murine models that mimic the human condition have been developed and are now widely used to investigate AAA pathogenesis. Cell- or pharmaceutical-based therapeutics designed to prevent AAA expansion are currently being evaluated with these animal models, but more minimally invasive strategies for delivery could improve their clinical translation. The purpose of this study was to investigate the use of self-assembling type I collagen oligomers as an injectable therapeutic delivery vehicle in mice. Here we show the success and reliability of a para-aortic, ultrasound-guided technique for injecting quickly-polymerizing collagen oligomer solutions into mice to form a collagen-fibril matrix at body temperature. A commonly used infrarenal mouse AAA model was used to determine the target location of these collagen injections. Ultrasound-guided, closed-abdominal injections supported consistent delivery of collagen to the area surrounding the infrarenal abdominal aorta halfway between the right renal artery and aortic trifurcation into the iliac and tail arteries. This minimally invasive approach yielded outcomes similar to open-abdominal injections into the same region. Histological analysis on tissue removed on day 14 post-operatively showed minimal in vivo degradation of the self-assembled fibrillar collagen and the majority of implants experienced minimal inflammation and cell invasion, further confirming this material's potential as a method for delivering therapeutics. Finally, we showed that the typical length and position of this infrarenal AAA model was statistically similar to the length and targeted location of the injected collagen, increasing its feasibility as a localized therapeutic delivery vehicle. Future preclinical and clinical studies are needed to determine if specific therapeutics incorporated into the self-assembling type I collagen matrix described here can be delivered near the aorta and locally limit AAA expansion.