The role of the plasminogen system in bone resorption in vitro.

The plasminogen/plasmin proteolytic cascade plays an important role in extracellular matrix remodeling. The presence of the two plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA), and their inhibitor type 1 (PAI-1) in bone cells, suggests a role in one or more aspects of bone resorption such as osteoclast formation, mineral dissolution, and degradation of the organic matrix. These different processes were assayed in vitro using cells derived from mice with either tPA (tPA-/-), uPA (uPA-/-), PAI-1 (PAI-1-/-) inactivation or with a combined inactivation (tPA-/-:uPA-/-) and compared with wild-type mice (WT). First, osteoclast formation, assessed by investigating the number and characteristics of tartrate-resistant acid phosphatase-positive multinucleated cells formed in cocultures of primary osteoblasts and bone marrow cells treated with 1alpha,25-dihydroxyvitamin D3, was not different between the different cell types. Second, dentine resorption, an assay for osteoclast activity, was not affected by the combined deficiency of both tPA and uPA. Finally, the ability to degrade nonmineralized bone-like matrix was however, significantly reduced in tPA-/-:uPA-/- cells compared with WT cells (28.1 +/- 0.6%, n = 6 vs. 56.4 +/- 3.1%, n = 6, respectively, p < 0.0001). Surprisingly, collagen proteolysis by bone cells was not dependent on the presence of plasmin as suggested by degradation assays performed on type I 3H-collagen films. Taken together, these data suggest that the plasminogen activator/plasmin system is not required for osteoclast formation, nor for the resorption of the mineral phase, but is involved in the removal of noncollagenous proteins present in the nonmineralized bone matrix.     

Plasminogen activators are involved in the degradation of bone by osteoclasts

Osteoclastic bone degradation depends on the activity of several proteolytic enzymes, in particular to those belonging to the classes of cysteine proteinases and matrix metalloproteinases (MMPs). Yet, several findings suggest that the two types of plasminogen activators (PA), the tissue- and urokinase-type PA (tPA and uPA, respectively) are also involved in this process. To investigate the involvement of these enzymes in osteoclast-mediated bone matrix digestion, we analyzed bone explants of mice that were deficient for both tPA and uPA and compared them to wild type mice. The number of osteoclasts as well as their ultrastructural appearance was similar for both genotypes. Next, calvarial and metatarsal bone explants were cultured for 6 or 24 h in the presence of selective inhibitors of cysteine proteinases or MMPs and the effect on osteoclast-mediated bone matrix degradation was assessed. Inhibition of the activity of cysteine proteinases in explants of control mice resulted in massive areas of non-digested demineralized bone matrix adjacent to the ruffled border of osteoclasts, an effect already maximal after 6 h. However, at that time point these demineralized areas were not observed in bone explants from uPA/tPA deficient mice. After prolonged culturing (24 h), a comparable amount of demineralized bone matrix adjacent to actively resorbing osteoclasts was observed in the two genotypes, suggesting that degradation was delayed in uPA/tPA deficient bones. The activity of cysteine proteinases as assessed in bone extracts, proved to be higher in extracts from uPA/tPA(-/-) bones. Immunolocalization of the integrin alpha(v)beta(3) of in vitro generated osteoclasts demonstrated a more diffuse labeling of osteoclasts derived from uPA/tPA(-/-) mice. Taken together, our data indicate that the PAs play a hitherto unrecognized role in osteoclast-mediated bone digestion. The present findings suggest that the PAs are involved in the initial steps of bone degradation, probably by a proper integrin-dependent attachment to bone.     

Tissue-type plasminogen activator modulates inflammatory responses and renal function in ischemia reperfusion injury

Acute renal failure is often the result of ischemia-reperfusion (I/R) injury. Neutrophil influx is an important damaging event in I/R. Tissue-type plasminogen activator (tPA) not only is a major fibrinolytic agent but also is involved in inflammatory processes. A distinct upregulation of tPA after I/R, with de novo tPA production by proximal renal tubules, was found. For investigating the role of tPA in I/R, renal ischemia was induced in tPA-/- and wild-type (WT) mice by clamping both renal arteries for 35 min followed by reperfusion. Mice were killed 1, 5, and 10 d after reperfusion. After 1 d, tPA-/- mice displayed significantly less neutrophil influx into the interstitial area compared with WT mice. In addition, tPA-/- mice showed quicker recovery of renal function than WT mice. The protocol was repeated after injection of tPA-antisense oligonucleotides into WT mice, leading to even more explicit results: Antisense-treated mice showed less histologic damage, better renal function, and less neutrophil influx than control mice. Surprising, complement C3 concentration, levels of proinflammatory cytokines and chemokines, intercellular adhesion molecule-1 expression, and matrix metalloproteinase activity were similar in WT and tPA-/- mice. Plasmin activity levels in WT and tPA-/- kidneys were also comparable, indicating that tPA influences neutrophil influx into ischemic renal tissue independent from plasmin generation. This study shows that targeting tPA could be of therapeutic importance in treating I/R injury by diminishing neutrophil influx and preserving renal function.     

Impaired fibrinolysis and traumatic brain injury in mice

Traumatic brain injury (TBI) has been associated with intravascular coagulation, which may be a result of thromboplastin released following brain injury. Clots thus formed are lysed by plasmin, which is activated by tissue-type and urokinase-type plasminogen activators (uPA). To evaluate the association between traumatic intravascular coagulation and post-traumatic outcome, uPA knockout (uPA-/-) transgenic mice (n=12) or wild-type littermates (WT; n=12) were anesthetized and subjected to controlled cortical impact (CCI) brain injury. A second group of uPA-/- (n=12) and WT mice (n=12) were subjected to sham injury. Motor function was assessed over 2 weeks using the composite neuroscore test and cognition (learning) was assessed with the Morris Water Maze (MWM) at 2 weeks post-injury, whereupon the animals were sacrificed for cortical lesion volume analysis. Motor function was significantly worse in the brain-injured uPA-/- mice when compared to brain-injured WT mice at 48 h (p<0.05) and one week post-injury (p<0.05). These differences resolved by 2 weeks post-injury. There was no significant difference in post-injury cognitive function between uPA-/- mice and WT mice. However, at 2 weeks post-injury, the brain-injured uPA-/- had a significantly larger volume of cortical tissue loss than their WT counterparts (p<0.05). These results demonstrate that the absence of uPA in mice aggravates acute motor deficit and exacerbates cortical tissue loss following CCI brain injury, and suggests a neuroprotective role of the fibrinolytic process following TBI.     

Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling

Urokinase plasminogen activator (uPA) regulates a proteolytic cascade of extracellular matrix degradation that functions in tissue development and tissue repair. The development and remodeling of the skeletal extracellular matrix during wound healing suggests that uPA might regulate bone development and repair. To determine whether uPA functions regulate bone development and repair, we examined the basal skeletal phenotype and endochondral bone fracture repair in uPA-deficient mice. The skeletal phenotype of uPA knockout mice was compared with that of control mice under basal conditions by dual-energy X-ray absorptiometry and micro-CT analysis, and during femur fracture repair by micro-CT and histological examination of the fracture callus. No effects of uPA gene deficiency were observed in the basal skeletal phenotype of the whole body or the femur. However, uPA gene deficiency resulted in increased fracture callus cartilage abundance during femur fracture repair at 14 days healing. The increase in cartilage corresponded to reduced tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts in the uPA knockout fracture callus at this time, consistent with impaired osteoclast-mediated remodeling of the fracture cartilage. CD31 staining was reduced in the knockout fracture tissues at this time, suggesting that angiogenesis was also reduced. Osteoclasts also colocalized with CD31 expression in the endothelial cells of the fracture tissues during callus remodeling. These results indicate that uPA promotes remodeling of the fracture cartilage by osteoclasts that are associated with angiogenesis and suggest that uPA promotes angiogenesis and remodeling of the fracture cartilage at this time of bone fracture repair.     

纤维蛋白对肾小球内皮细胞表达纤溶酶原激活物及纤溶酶原激活物抑制物的作用

目的：观察体外培养人肾小球内皮细胞（GEC）表面原位形成的纤维蛋白对GEC表达纤溶酶原激活物及纤溶酶原激活物抑制物（PA／PAI）的影响。方法：应用逆转录聚合酶链反应（RT－PCR）,酶谱分析法与反向酶谱法分别在基因转录水平与蛋白质活性水平上检测纤维蛋白对GEC表达tPA,uPA gn PAI-1r 作用,纤维蛋白平板法检测纤维蛋白对GEC PA／PAI系统的综合效应,结果：纤维蛋白能够明显促进tPA,uPA与PAI－1的mRNA表达上调,无血清RPMI 1640培养下的GEC几乎检测不到PAI知性,但可检测到PAI－1的活性。纤维蛋白能够浓度依赖性刺激GEC tPA与uPA活性增加以及PAI01的活性增加,呈浓度依赖性与时间依赖性,相同剂量的纤维蛋白原与纤维蛋白的作用相似,放线菌酮与放线菌素D均可抑制纤维蛋白上调GEC表达tPA,uPA与PAI的作用,纤维蛋白平板法显示,纤维蛋白对GEC PA／PAI系统的综合效应是以升高PA活性为主,其活性能够被抑肽酶完全阻断。结论：肾脏局部毛细血[管内沉积的纤维蛋白可能通过对GEC PA／PAI系统的调节发挥其病理作用。     

Relationships between Activators and Inhibitors of Plasminogen, and the Progression of Small Abdominal Aortic Aneurysms

OBJECTIVE: plasmin is a common activator of the known proteolytic systems involved in the aneurysmal degradation, and is reported to be associated with the expansion of abdominal aortic aneurysms (AAA). The aim of this study was to study the activating pathways of plasminogen as predictors of the progression of AAA. MATERIALS AND METHODS: one hundred and twelve of 122 male patients with a small AAA (def.: +3cm) were interviewed, examined, had blood samples taken at diagnosis, and scanned annually for 1-5 years (mean 3.5 years), and referred for surgery if the AAA exceeded 5cm in diameter.A random sample of 70 of the 112 cases had plasma levels of urokinase-like-plasminogen activator (uPA), tissue-type-plasminogen activator (tPA), plasminogen-activator-inhibitor-1 (PAI-1), macrophage inhibiting factor (MIF), tumour-growth-factor-beta1 (TGF-beta1), homocysteine, and serum levels of IgA-antibodies against Chlamydia pneumoniae (IgA-CP) and Cotinine (a nicotine metabolite) measured. Spearmans correlation analysis was used for statistics. RESULTS: the annual expansion rate correlated positively with tPA, IgA-CP and S-Cotinine; r =0.37 (p=0.002), 0.29 (p=0.006) and 0.24 (p=0.038), while PAI1, uPA, TGF-beta1, homocysteine, and MIF did not. S-Cotinine did also correlate positively with tPA, r=0.24 (p=0.049). CONCLUSION: the aortic matrix degradation in AAA may be partly caused by an activation of plasminogen by tPA, but apparently not by uPA, which usually dominates matrix degradation. Smoking seems to be a factor for this pathway, while the pathways of IgA-CP and MIF, a new marker of aneurysmal progression, seem different. The latter observations suggest that other proteolytic pathways are involved in the aortic wall degradation in AAA.     

Cyclosporine withdrawal and mycophenolate mofetil treatment effects on the progression of chronic cyclosporine nephrotoxicity

BACKGROUND: Dysregulated expression of diverse proteases and their specific inhibitors is critical for the increase in extracellular matrix accumulation that accompanies chronic inflammatory and sclerotic processes within the renal mesangium. Within the activating cascade of several proteases, the plasminogen system plays an important role. METHODS: We tested for modulatory effects of the nitric oxide (NO) donors S-nitroso-N-acetyl-D,L-penicillamine and DETA-NONOate, and the superoxide-generating system hypoxanthine/xanthine oxidase (HXXO) on the expression and activity of tissue plasminogen activator (tPA) by ELISA and Northern blotting. RESULTS: Interleukin-1beta (IL-1beta)-induced tPA and plasminogen activator inhibitor (PAI)-1 mRNA and supernatant tPA antigen were significantly inhibited by both NO donors, which resulted in a net decrease in the IL-1beta-evoked tPA enzyme activity in the conditioned media. Addition of the NO-synthase inhibitor N-monomethyl l-arginine markedly increased the cytokine-triggered tPA- and PAI-1 mRNA levels, respectively. In contrast, HXXO caused a marked amplification of the IL-1beta-induced steady-state tPA mRNA level and tPA enzyme activity that was blocked by catalase. Since MnTBAP, a superoxide dismutase mimetic, had no effects on the amplification of mRNA levels, we suggest that H2O2 is the candidate reactive oxygen species (ROS) responsible for the potentiation of IL-1beta-triggered tPA and PAI-1 expression. CONCLUSIONS: The temporal relationship between NO and ROS generation is a critical step in the modulation of tPA and PAI-1 expression in mesangial cells and may account for a dysregulation of matrix turnover during inflammatory processes in the renal mesangium.     

Action of IL‐1β during fracture healing

After bone injury, developmental processes such as endochondral and intramembranous ossification are recapitulated as the skeleton regenerates. In contrast to development, skeletal healing involves inflammation. During the early stages of healing a variety of inflammatory cells infiltrate the injured site, debride the wound, and stimulate the repair process. Little is known about the inflammatory process during bone repair. In this work, we examined the effect of a pro‐inflammatory cytokine, Interleukin‐1 beta (IL‐1β), on osteoblast and stem cell differentiation and on intramembranous and endochondral ossification, because IL‐1β exerts effects on skeletal homeostasis and is upregulated in response to fracture. We determined that IL‐1β stimulated proliferation of osteoblasts and production of mineralized bone matrix, but suppressed proliferation and inhibited differentiation of bone marrow derived MSCs. We next performed loss‐ and gain‐of‐function experiments to determine if altering IL‐1β signaling affects fracture healing. We did not detect any differences in callus, cartilage, and bone matrix production during healing of nonstabilized or stabilized fractures in mice that lacked the IL‐1β receptor compared to wild‐type animals. We observed subtle alterations in the healing process after administering IL‐1β during the early phases of repair. At day 10 after injury, the ratio of cartilage to callus was increased, and by day 14, the proportion of cartilage to total callus and to bone was reduced. These changes could reflect a slight acceleration of endochondral ossification, or direct effects on cartilage and bone formation. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:778–784, 2010     

Tissue and urokinase plasminogen activators in bone tissue and their regulation by parathyroid hormone.

The identification of the plasminogen activator (PA) types present in bone and the regulation of their activity by parathyroid hormone (PTH) were investigated in cultures of fetal mouse calvariae with the use of either a chromogenic substrate or a zymographic assay. PA was detected essentially in the tissue extracts of the explanted bones, with only 1-2% of the total activity released in the surrounding culture media. From their electrophoretic behavior compared to PAs of other mouse tissues and from their response to a specific antibody raised against the tissue type PA (tPA), two major molecular species, of 70 and 48 kD were identified as tPA and urokinase (uPA), respectively, a third minor species of 105 kD being likely to correspond to complexes between tPA and an inhibitor; the culture fluids, moreover, contained enzymatically active degradation products of uPA of 42 and 29 kD. The PA activity of the bone extracts was only minimally affected by the addition of fibrinogen fragments to the chromogenic assays. PTH induced bone resorption and stimulated in parallel the accumulation of PA in the tissue; other bone-resorbing agents, 1,25-dihydroxyvitamin D3 and prostaglandin E2, had similar effects. Densitometric scanning of the zymograms of the bone extracts indicated that PTH stimulated only the production of tPA and had no effect on that of uPA. However, PTH also enhanced the release of uPA (both the 48 kD and the 29 kD forms) from the bones into the media. Although inhibiting bone resorption, calcitonin had no effect on the PTH-induced accumulation of PA in bone or on the release of tPA, but it prevented the PTH-induced accumulation of 29 kD uPA in the culture fluids. Thus these studies support the view that tPA and possibly also uPA may have a role in the physiology of bone; the nature of this role remains to be elucidated, however.     

Twisted gastrulation, a bone morphogenetic protein agonist/antagonist, is not required for post-natal skeletal function

Twisted gastrulation (Tsg) is a secreted glycoprotein that binds bone morphogenetic proteins (BMP)-2 and -4 and can display both BMP agonist and antagonist functions. Tsg promotes BMP-mediated endochondral ossification, but its activity in adult bone is not known. We created tsg null mice and examined the consequences of the tsg deletion on the skeleton in vivo and on osteoblast function in vitro. Analysis of the skeletal phenotype of 4-week-old tsg null mice revealed a 40% decrease in trabecular bone volume, but osteoblast and osteoclast number, and bone formation and resorption were not affected. The phenotype was transient, and at 7 weeks of age tsg null mice were not different from control wild-type mice. The decreased trabecular bone is congruent with a defect in endochondral bone formation. In osteoblasts isolated from tsg null mice, tsg gene inactivation decreased the BMP-2 stimulatory effects on osteocalcin expression and alkaline phosphatase activity, indicating that in the bone microenvironment endogenous Tsg enhances BMP activity. Accordingly, tsg null cells displayed impaired BMP signaling. These results were confirmed by Tsg down-regulation in primary osteoblasts from wild-type mice using RNA interference. In conclusion, endogenous Tsg is required for normal BMP activity in osteoblastic cells in vitro, but it plays a minor role in the regulation of adult bone homeostasis in vivo.     

Epiphyseal Chondrocyte Secondary Ossification Centers Require Thyroid Hormone Activation of Indian Hedgehog and Osterix Signaling

Thyroid hormones (THs) are known to regulate endochondral ossification during skeletal development via acting directly in chondrocytes and osteoblasts. In this study, we focused on TH effects on the secondary ossification center (SOC) because the time of appearance of SOCs in several species coincides with the time when peak levels of TH are attained. Accordingly, micro–computed tomography (µCT) evaluation of femurs and tibias at day 21 in TH‐deficient and control mice revealed that endochondral ossification of SOCs is severely compromised owing to TH deficiency and that TH treatment for 10 days completely rescued this phenotype. Staining of cartilage and bone in the epiphysis revealed that whereas all of the cartilage is converted into bone in the prepubertal control mice, this conversion failed to occur in the TH‐deficient mice. Immunohistochemistry studies revealed that TH treatment of thyroid stimulating hormone receptor mutant (Tshr^?/?) mice induced expression of Indian hedgehog (Ihh) and Osx in type 2 collagen (Col2)‐expressing chondrocytes in the SOC at day 7, which subsequently differentiate into type 10 collagen (Col10)/osteocalcin‐expressing chondro/osteoblasts at day 10. Consistent with these data, treatment of tibia cultures from 3‐day‐old mice with 10 ng/mL TH increased expression of Osx, Col10, alkaline phosphatase (ALP), and osteocalcin in the epiphysis by sixfold to 60‐fold. Furthermore, knockdown of the TH‐induced increase in Osx expression using lentiviral small hairpin RNA (shRNA) significantly blocked TH‐induced ALP and osteocalcin expression in chondrocytes. Treatment of chondrogenic cells with an Ihh inhibitor abolished chondro/osteoblast differentiation and SOC formation. Our findings indicate that TH regulates the SOC initiation and progression via differentiating chondrocytes into bone matrix–producing osteoblasts by stimulating Ihh and Osx expression in chondrocytes. © 2014 American Society for Bone and Mineral Research.     

Overexpression of Smurf2 Stimulates Endochondral Ossification Through Upregulation of β‐Catenin

Ectopic expression of Smurf2 in chondrocytes and perichondrial cells accelerated endochondral ossification by stimulating chondrocyte maturation and osteoblast development through upregulation of β‐catenin in Col2a1‐Smurf2 embryos. The mechanism underlying Smurf2‐mediated morphological changes during embryonic development may provide new mechanistic insights and potential targets for prevention and treatment of human osteoarthritis. Introduction : Our recent finding that adult Col2a1‐Smurf2 mice have an osteoarthritis‐like phenotype in knee joints prompted us to examine the role of Smurf2 in the regulation of chondrocyte maturation and osteoblast differentiation during embryonic endochondral ossification. Materials and Methods : We analyzed gene expression and morphological changes in developing limbs by immunofluorescence, immunohistochemistry, Western blot, skeletal preparation, and histology. A series of markers for chondrocyte maturation and osteoblast differentiation in developing limbs were examined by in situ hybridization. Results : Ectopic overexpression of Smurf2 driven by the Col2a1 promoter was detected in chondrocytes and in the perichondrium/periosteum of 16.5 dpc transgenic limbs. Ectopic Smurf2 expression in cells of the chondrogenic lineage inhibited chondrocyte differentiation and stimulated maturation; ectopic Smurf2 in cells of the osteoblastic lineage stimulated osteoblast differentiation. Mechanistically, this could be caused by a dramatic increase in the expression of β‐catenin protein levels in the chondrocytes and perichondrial/periosteal cells of the Col2a1‐Smurf2 limbs. Conclusions : Ectopic expression of Smurf2 driven by the Col2a1 promoter accelerated the process of endochondral ossification including chondrocyte maturation and osteoblast differentiation through upregulation of β‐catenin, suggesting a possible mechanism for development of osteoarthritis seen in these mice.     

7/8肾切除大鼠凝血纤溶系统的变化及干预治疗的影响

目的：探讨肾小球硬化及小管间质纤维化过程中肾组织纤溶酶原激活物（PA）和PA抑制物（PAI）－1蛋白表达及干预治疗的影响。方法：7／8肾切除肾功能衰竭大鼠为实验动物模型,随机分为未治疗组和治疗组,观察12周后各组大鼠血尿各项生化指标、尿PA活性及残肾组织常规病理和用免疫组织化学染色定性定量评价残肾组织型PA（tPA)、尿激酶型（uPA)、PAI－1蛋白表达。结果：未治疗组大鼠肾功能进行性丧失,尿PA活性下降,肾组织PAI－1表达增高,而tPA、uPA表达下降,残肾组织出现肾小球硬化和间质纤维化。治疗组大鼠残肾组织tPA、uPA蛋白表达增加,PAI－1表达下降。尿PA活性增加,肾功能改善。结论：水蛭治疗组、苯那普利治疗组及联合治疗组都可以通过改善7／8肾切除大鼠PA／PAI－1系统的紊乱而延缓肾小球硬化和间质纤维化病变的进展。     

Inhibition of growth plate angiogenesis and endochondral ossification with diminished expression of MMP-13 in hypertrophic chondrocytes in FGF-2-treated rats

Fibroblast growth factors (FGFs)/fibroblast growth factor receptor-3 signaling interferes with endochondral bone growth. However, the exact mechanisms by which FGFs inhibit endochondral ossification remain to be elucidated. In the present study, we utilized immunohistochemical techniques to clarify the effects of FGF-2 on the proximal tibial growth plate cartilage, when injected systemically into growing rats. In the FGF-2-treated rats, the growth plate was obviously thickened and, in the lowermost part, the hypertrophic chondrocytes were flattened, with an irregular arrangement. The connection of the cartilage columns and trabecular bone was disrupted. FGF-2 treatment stimulated the proliferation of chondrocytes and permitted their differentiation, but inhibited vascular invasion and resorption of the cartilage matrix. Expression of matrix metalloproteinase-13 (MMP-13) was detected in the chondrocytes in the last row of the hypertrophic zone of the growth plate in control animals. The immunoreactivity of MMP-13 was diminished in the regions where endochondral ossification was disturbed in the FGF-2-treated rats. Because MMP-13 has potent proteolytic activity on cartilage components, the FGF-2 signal may inhibit angiogenesis and endochondral ossification of the growth plate by the suppression of MMP-13 expression in hypertrophic chondrocytes. Received: March 17, 2001 / Accepted: November 16, 2001     

Effect of tissue plasminogen activator on vascular smooth muscle cells

OBJECTIVE: Engineered overexpression of tissue plasminogen activator (tPA) in vascular cells has been proposed as a means to decrease intravascular thrombosis; however, tPA gene transfer has augmented intimal hyperplasia in vivo in some studies. The purpose of this study was to define in vitro the effect of tPA gene transfer on smooth muscle cells (SMCs). METHODS: Human SMCs were retrovirally transduced with the tPA gene (SMCs/tPA). RESULTS: In the absence of plasminogen, no statistical differences in proliferation, migration, and morphology were observed between SMCs/tPA and SMCs. In the presence of plasminogen, many differences became apparent. Matrix metalloproteinase-2 (MMP-2) activation was 10-fold higher in SMCs/tPA than in SMCs. This activation was inhibited by aprotinin, a plasmin inhibitor. Collagen degradation increased sevenfold in SMCs/tPA. SMCs/tPA contracted dramatically in the presence of plasminogen. This cell contraction, indicative of extracellular matrix degradation, was blocked by aprotinin and partially inhibited by MMP inhibitors. SMC/tPA-conditioned medium induced significantly more SMC proliferation. The migration of SMCs/tPA through a porous membrane significantly exceeded untransduced SMCs. CONCLUSIONS: Over-expression of tPA in SMCs results in increased extracellular matrix degradation and can promote cell proliferation and migration. This effect is mediated via plasmin, which further activates MMP-2. CLINICAL RELEVANCE: TPA has been clinically used as a thrombolytic agent in the treatment of acute thrombotic disorders. Transferring the tPA gene into vascular cells as a strategy of gene therapy has been proposed to enhance fibrinolytic capability and therefore inhibit thrombosis and restenosis after vascular interventions. The mechanism(s) by which tPA affects SMC proliferation and vascular remodeling has not been thoroughly characterized. This study unveils the relationship between thrombolytic activity and intimal hyperplasia by showing how the elevated expression of tPA affects the vascular remodeling. This study underscores that the overexpression of an enzyme thought beneficial to blood flow can potentially compromise blood flow by altering the biology of the cell engineered to express it. The results are important to the rational engineering of bioactive grafts with better patency. A new strategy to enhance the thrombolytic ability of a vascular surface without inducing excessive neointimal hyperplasia is proposed.     

The matrix of endochondral bone differs from the matrix of intramembranous bone

Summary Osseous tissue develops via two distinctly different processes: endochondral (EC) ossification and intramembranous (IM) ossification. The present study tests the hypothesis that each type of osseous tissue contains unique inducing factors for the promotion of cartilage and bone development. Previous work suggests that subcutaneous implants of demineralized EC and IM bone matrices both induce endochondral ossification. Thus, it concludes that the bone growth promotion properties of the respective matrices are very similar. As it was unclear to us why EC and IM bone powders should possess identical osteoinductive properties, we attempted to reproduce these results. We implanted EC (femoral) demineralized bone matrix (DBM), IM (frontal) DBM, or a mixture of the two into the ventral thoracic subcutaneous tissue of 12 to 15-week-old male Sprague Dawley rats. Morphological and radiolabeling techniques in this study demonstrated that implants of EC bone matrix induce bone formation via EC ossification in contrast to implants of IM bone matrix which do not induce EC ossification. Our findings suggest that the matrix of EC bone differs qualitatively from the matrix of IM bone due to their respective abilities to induced cartilage and/or bone formation. These observations differ from those previously reported possibly because our IM DBM preparations were not contaminated with tissues of endochondral origin. In current clinical practice, EC DBM allografts are often used to induce new bone formation in defects involving both IM and EC bone. We conclude that there may be clinical settings in which it would be more appropriate to replace bone originally formed via IM ossification with IM DBM rather than EC DBM.