Perlecan domain I-conjugated, hyaluronic acid-based hydrogel particles for enhanced chondrogenic differentiation via BMP-2 release

We have developed a biomimetic growth factor delivery system that effectively stimulates the chondrogenic differentiation of the cultured mesenchymal stem cells via the controlled presentation of bone morphogenetic protein-2 (BMP-2). Hyaluronic acid (HA)-based, microscopic hydrogel particles (HGPs) with inherent nanopores and defined functional groups were synthesized by an inverse emulsion polymerization technique. Recombinantly produced, heparan sulfate (HS)-bearing perlecan domain I (PlnDI) was covalently immobilized to HA HGPs (HGP–P₁) via a flexible poly(ethylene glycol) (PEG) linker through the lysine amines in the core protein of PlnDI employing reductive amination. Compared to HGP without PlnDI, HGP–P₁ exhibited significantly (p < 0.05) higher BMP-2 binding capacity and distinctly different BMP-2 release kinetics. Heparitinase treatment increased the amount of BMP-2 released from HGP–P₁, confirming the HS-dependent BMP-2 binding. While BMP-2 was released from HGPs with a distinct burst release followed by a minimal cumulative release, its release from HGP–P₁ exhibited a minimal burst release followed by linear release kinetics over 15 days. The bioactivity of the hydrogel particles was evaluated using micromass culture of multipotent mesenchymal stem cells (MSCs), and the chondrogenic differentiation was assessed by the production of glycosaminoglycan, aggrecan and collagen type II. Our results revealed that BMP-2 loaded HGP–P₁ stimulates more robust cartilage specific ECM production as compared to BMP-2 loaded HGP, due to the ability of HGP–P₁ to potentiate BMP-2 and modulate its release with a near zero-order release kinetics. The PlnDI-conjugated, HA HGPs provide an improved BMP-2 delivery system for stimulating chondrogenic differentiation in vitro, with potential therapeutic application for cartilage repair and regeneration.     

Development of the coronary arteries in a murine model of transposition of great arteries

Transposition of great arteries in humans is associated with a wide spectrum of coronary artery patterns. However, no information is available about how this pattern diversity develops. We have studied the development of the coronary arteries in mouse embryos with a targeted mutation of perlecan, a mutation that leads to ventriculo-arterial discordance and complete transposition in about 70% of the embryos. The perlecan-deficient embryos bearing complete transposition showed a coronary artery pattern consisting of right and left coronary arteries arising from the morphologically dorsal and ventral sinuses of Valsalva, respectively. The left coronary artery gives rise to a large septal artery and runs along the ventral margin of the pulmonary root. In the earliest embryos where transposition could be confirmed (12.5 d post coitum), a dense subepicardial vascular plexus is located in this ventral margin. In wild-type mice, however, capillaries are very scarce on the ventral surface of the pulmonary root and the left coronary artery runs dorsally to this root. We suggest that the establishment of the diverse coronary artery patterns is determined by the anatomical arrangement and the capillary density of the peritruncal vascular plexus, a plexus that spreads from the atrio-ventricular groove and grows around the aortic or pulmonary roots depending on the degree of the short-axis aortopulmonary rotation. This simple model, based on very few assumptions, might explain all the observed variation of the coronary artery patterns in humans with transposition, as well as our observations on the perlecan-deficient and the normal mice.     

Wert der Basalmembrandarstellung in der Diagnostik invasiver Karzinome

Zusammenfassung Die Destruktion der epithelialen Basalmembran gilt weithin als wichtiges Kriterium für malignes, invasives Wachstum. Der diagnostische Wert dieser Beobachtung wird jedoch eingeschr?nkt durch die Beobachtung, da? die Basalmembran auch bei bestimmten nichtinvasiven Epithelver?nderungen defekt sein kann, ebenso wie z.B. bei hochdifferenzierten Plattenepithelkarzinomen eine intakte Basalmembran durch den Nachweis einzelner Komponenten vorgespielt werden kann. Dennoch kann eine immunhistochemische Basalmembranlokalisation in bestimmten F?llen von diagnostischem Nutzen sein. Dies gilt insbesondere für invasive (duktale) Mammakarzinome, die in aller Regel einen kompletten Basalmembranverlust zeigen, so da? hierdurch im Einzelfall eine Unterscheidung zwischen sklerosierender Adenose und inasivem Karzinom und zwischen tubul?rem Karzinom und nichtmaligner tubul?rer Epithelproliferation m?glich wird.      

Comparative immunolocalisation of perlecan, heparan sulphate, fibroblast growth factor-18, and fibroblast growth factor receptor-3 and their prospective roles in chondrogenic and osteogenic development of the human foetal spine

Purpose

A comparative immunolocalisation study of perlecan, HS, FGF-18 and FGFR-3 in the 12–20-week gestational age human foetal spine was undertaken to identify spatiotemporal associations between these components to provide insights into prospective roles in spinal development.

Methods

Comparative immunolocalisations of matrix and cell associated components in Histochoice-fixed paraffin-embedded human foetal spinal tissues.

Results

The 12–14-week-old human foetal spine was a predominantly cartilaginous structure with the discs displaying a relative paucity of proteoglycan compared to the adjacent cartilaginous vertebral rudiments, notochordal remnants were also observed. HS and perlecan had a widespread distribution throughout the spine at 12 weeks, however, FGF-18 was only localised to the outer AF margins and hypertrophic cell condensations in the vertebral bodies. This contrasted with HS distributions at 14–20 weeks, which were prominent in the developing intervertebral disc (IVD). Ossification centres were also evident centrally within the vertebral rudiments surrounded by small columns of hypertrophic chondrocytes which expressed FGFR-3 and FGF-18 and upregulated levels of perlecan. FGF-18 also had a prominent localisation pattern in the developing IVD and the cartilaginous endplate while FGFR-3 was expressed throughout the disc interspace. This suggested roles for perlecan, FGF-18 and FGFR-3 in chondrogenic and osteogenic events which drive discal development and ossification of the vertebral bodies.

Conclusions

The above data supported a role for FGF-18 in discal development and in the terminal osteogenic differentiation of chondroprogenitor cell populations, which promote vertebral ossification during spinal development.     

Perlecan (heparan sulfate proteoglycan) gene expression reflected in the characteristic histological architecture of salivary adenoid cystic carcinoma

In order to determine the role of the basement membrane-type heparan sulfate proteoglycan (HSPG), known as perlecan, in the formation of the characteristic cribriform structures of salivary adenoid cystic carcinomas, the mode of expression of mRNA for the core protein of HSPG was investigated by using in situ hybridization (ISH) both in surgical specimens and in a cell system (ACC3) of adenoid cystic carcinomas. In the surgical specimens, the mRNA for the HSPG core was more intensely expressed in solid tumor cell nests, especially in smaller ones. Within the nests, the signals were detected almost exclusively in cuboidal cells forming small pseudocysts. In contrast, signals were absent in flat cells forming large pseudocysts or in carcinoma cell nests attaching to the peripheral nerves or blood vessels. In normal salivary gland tissues, myoepithelial cells expressed the mRNA at a high level, but acinar and duct epithelial cells did not. In the time-course experiment of ACC3 cells, signals for HSPG core increased with time and reached the maximum on day 4, decreasing thereafter in a culture condition in which cells reached confluence in a week. The results indicate that HSPG is biosynthesized by adenoid cystic carcinoma cells which are in the proliferation phase, and that tumor cells producing HSPG tend to form initial structures of stromal pseudocysts. Received: 29 September 1999 / Accepted: 15 February 2000     

What maintains the high intra-follicular estradiol concentration in pre-ovulatory follicles?

Purpose

The purpose of the study was to establish the mechanism by which the estrogen concentration difference between the follicular fluid and the serum is maintained.

Methods

We used dialysis membrane with a pore size of <3 KD to characterize the estrogen-binding capacity of the follicular fluid. We performed PCR, western blot, and ELISA on luteinized granulosa cells to determine if sex hormone-binding globulin (SHBG) is produced by granulosa cells, and finally we used affinity columns and mass spectrometry to identify the estrogen-binding protein in the follicular fluid.

Results

We found that a significant estrogen concentration difference is maintained in a cell-free system and is lost with proteolysis of the follicular fluid proteins. Luteinized granulosa cells are likely not a source of SHBG, as we were not able to detect expression of SHBG in these cells. Perlecan was the most highly enriched follicular fluid protein in the affinity columns.

Conclusions

We were able to identify perlecan as the most likely candidate for the major estrogen-binding protein in the follicular fluid.

     

PRELP,collagen, and a theory of Hutchinson-Gilford progeria

Proline/arginine-rich end leucine-rich repeat protein (PRELP) a small leucine-rich proteoglycan (SLRP), binds type I collagen to basement membranes and type II collagen to cartilage. Evidence for lack of binding of collagen in basement membranes and cartilage of Hutchinson-Gilford progeria (HGP) cases suggests PRELP involvement in that disease. PRELP deficiency is able to account for many symptoms of HGP. Moreover, PRELP also accounts for the fact that unlike many other collagen-related diseases, HGP symptoms are not congenital. The appearance of PRELP sometime after the third month of the birth, coincides with the appearance of HGP symptoms. Hutchinson-Gilford progeria has been diagnosed in twins with a chromosomal inversion at, or very near, the site of the PRELP gene.     

串珠素在喉癌细胞Hep-2中的表达

目的:研究串珠素(perlecan)在喉癌细胞Hep-2中的表达,探讨其与喉癌细胞的生长、浸润及转移的关系。方法:培养Hep-2和正常永生化表皮细胞系(Hacat),提取总细胞RNA,应用RT-PCR方法检测串珠素mRNA在Hep-2和Hacat细胞中的表达;应用免疫组织化学染色检测串珠素在Hep-2细胞和Hacat细胞中的表达。结果:串珠素mRNA在Hep-2细胞中高表达,平均相对积分吸收度(A)值为:0.831 0±0.050 4;串珠素mRNA在Hacat细胞中低表达,平均相对积分A值为:0.274 3±0.062 9。差异有统计学意义(t=19.684,P<0.01)。免疫组织化学染色表明串珠素在Hep-2细胞中高表达(A=0.255 7±0.028 7),在Hacat细胞中低表达(A=0.073 0±0.011 1),差异有统计学意义(t=26.982,P<0.01)。结论:串珠素与喉癌细胞的生长、浸润及转移有密切关系。     

串珠素反义cDNA质粒转染对喉癌细胞Hep-2增殖能力的影响

目的：研究串珠素反义cDNA质粒转染对喉癌细胞Hep-2增殖能力的影响。方法：利用阳离子脂质体作为载体，把重组真核表达载体串珠素反义cDNA质粒pAP转染喉癌细胞Hep-2。通过RT—PCR、Westernblot及MTT法检测转染后Hep-2细胞串珠素mRNA、蛋白质表达及细胞增殖水平，并观察转染后Hep-2细胞对碱性成纤维细胞生长因子（bFGF）的反应性。将Hep2细胞分3组：①未转染的Hep-2细胞组（WT组）；②空载体ph8Apr—neol转染组（neo组）；③串珠素反义cDNA质粒pAP转染组（pAP组）。结果：将质粒pAP成功地转入Hep2细胞，获得了稳定表达串珠素反义cDNA基因的细胞系。串珠素mRNA和蛋白质水平在pAP组低表达，在WT组和neo组高表达，均差异有统计学意义（均P〈O．01）。在0．1％FCS的RPMI1640培养基培养下，WT组、neo组和pAP组细胞的增殖率均降低，但pAP组细胞的增殖与WT组和neo组细胞相比明显减低；在0．1％FCS加1μg／L bFGF的RPMI1640培养液培养下．WT组和neo组细胞增殖接近正常，但pAP组细胞的增殖能力较弱。结论：串珠素反义cDNA质粒转染可以有效抑制喉癌细胞Hep2的增殖能力。     

妊娠期糖尿病胎盘病理与串珠素关系研究

目的:探讨妊娠期糖尿病胎盘的病理改变及其与串珠素表达的关系。方法:取妊娠期糖尿病及正常妊娠足月分娩胎盘各30例,石蜡切片,光镜下观察两组胎盘形态,免疫组织化学法标记胎盘组织中串珠素的表达水平。结果:病例组胎盘存在明显的干绒毛小动脉增生、绒毛成熟不良及毛细血管充盈现象,串珠素表达水平明显高于对照组,差异具有统计学意义(P0.05)。结论:妊娠期糖尿病导致胎盘血管病理改变与串珠素的表达水平增高有关。