Search

Currently the world is dealing with the third outbreak of the human-infecting coronavirus with potential lethal outcome, cause by a member of the Nidovirus family, the SARS-CoV-2. The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused the last worldwide pandemic. Successful development of vaccines highly contributed to reduce the severeness of the COVID-19 disease. To establish a control over the current and newly emerging coronaviruses of epidemic concern requires development of substances able to cure severely infected individuals and to prevent virus transmission. Here we present a therapeutic strategy targeting the SARS-CoV-2 RNA using antisense oligonucleotides (ASOs) and identify locked nucleic acid gapmers (LNA gapmers) potent to reduce by up to 96% the intracellular viral loadin vitro. Our results strongly suggest promise of our preselected ASOs for further development as therapeutic or prophylactic anti-viral agents.One sentence summaryASOs (LNA gapmers) targeting the SARS-CoV-2 RNA genome have been effective in viral RNA (load) reductionin vitro.

The durability of prosthetic arteriovenous (AV) grafts for hemodialysis access is low, predominantly due to stenotic lesions in the venous outflow tract and infectious complications. Tissue engineered blood vessels (TEBVs) might offer a tailor-made autologous alternative for prosthetic grafts. We have designed a method in which TEBVs are grown in vivo, by utilizing the foreign body response to subcutaneously implanted polymeric rods in goats, resulting in the formation of an autologous fibrocellular tissue capsule (TC). One month after implantation, the polymeric rod is extracted, whereupon TCs (length 6 cm, diameter 6.8 mm) were grafted as arteriovenous conduit between the carotid artery and jugular vein of the same goats. At time of grafting, the TCs were shown to have sufficient mechanical strength in terms of bursting pressure (2382 +/- 129 mmHg), and suture retention strength (SRS: 1.97 +/- 0.49 N). The AV grafts were harvested at 1 or 2 months after grafting. In an ex vivo whole blood perfusion system, the lumen of the vascular grafts was shown to be less thrombogenic compared to the initial TCs and ePTFE grafts. At 8 weeks after grafting, the entire graft was covered with an endothelial layer and abundant elastin expression was present throughout the graft. Patency at 1 and 2 months was comparable with ePTFE AV-grafts. In conclusion, we demonstrate the remodeling capacity of cellularized in vivo engineered TEBVs, and their potential as autologous alternative for prosthetic vascular grafts.

Summary Background Bone marrow-derived circulating CD34+ progenitor cells participate in remodeling and repair of the vasculature. Coexpression of the kinase-insert domain-containing receptor (KDR) has been proposed to identify the regenerative capacity. Recently, we provided evidence that the major fraction of circulating CD34+/KDR+ cells is not mobilized from bone marrow, but is generated at sites of vascular injury through interaction with platelets. Objectives To determine the relationship between platelet activation, the recruitment of naive CD34+ cells and the generation of CD34+/KDR+ progenitor cells in a broad range of (patho)physiologic conditions, a detailed meta-regression analysis was conducted. Methods/Results Twenty-eight conditions were found in which the numbers of CD34+ and/or CD34+/KDR+ cells and the levels of soluble P-selectin, as a marker for in vivo platelet activation, were documented. To combine heterogeneous data from 214 selected articles, results were standardized to a uniform scale by calculating standardized mean differences (SMDs) obtained from patient and control cohorts. Subsequently, a random-effects meta-regression analysis was performed on pooled SMDs. Conclusions Our systemic survey supports a model in which activated platelets are a determinant for mobilization of CD34+ cells from the bone marrow and the generation of CD34+/KDR+ cells in the circulation.

BackgroundWe need new biomarkers that can predict cardiovascular disease to improve both diagnosis and therapeutic strategies. The CIRCULATING CELLS study was designed to study the role of several cellular mediators of atherosclerosis as biomarkers of coronary artery disease (CAD). An objective and reproducible method for the quantification of CAD extension is required to establish relationships with these potential biomarkers. We sought to analyse the correlation of the SYNTAX score with known CAD risk factors to test it as a valid marker of CAD extension.Methods and resultsA subgroup of 279 patients (67.4% males) were included in our analysis. Main exclusion criteria were a history of previous percutaneous coronary intervention or surgical revascularisation that prevent an accurate assessment of the SS. Diabetes mellitus, smoking, renal insufficiency, body mass index and a history of CAD and myocardial infarction were all positively and strongly associated with a higher SYNTAX score after adjustment for the non-modifiable biological factors (age and sex). In the multivariate model, age and male sex, along with smoking and renal insufficiency, remain statistical significantly associated with the SYNTAX score.ConclusionIn a selected cohort of revascularisation-naive patients with CAD undergoing coronary angiography, non-modifiable cardiovascular risk factors such as advanced age, male sex, as well as smoking and renal failure were independently associated with CAD complexity assessed by the SYNTAX score. The SYNTAX score may be a valid marker of CAD extension to establish relationships with potential novel biomarkers of coronary atherosclerosis.Keywords: Coronary artery disease, Coronary angiography, Risk assessment, Risk factors

The transition from 2D to 3D cell culture is a first step towards more physiologically relevant in vitro cancer models. To adequately capture the complex tissue architectures observed in vivo, 3D microfluidic techniques incorporate and achieve long-term gradient stability, continuous perfusion and patterning of cancer cell layers as stratified co-cultures. We used a standardized high-throughput (n=40) microfluidic 3D tissue culture platform called the OrganoPlate® to generate precisely controlled gradients, without pumps, ideal for growing blood vessels and inducing controlled 3D angiogenic sprouting. The blood vessel is grown against an extracellular matrix (ECM) gel with cancer cells and is subsequently exposed to pro- and antiangiogenic compounds to direct sprouting towards 3D cancer cell clusters. Utilizing high-content confocal time-lapse imaging and analysis, angiogenic potential was measured in various cancer models. The exposed vasculature shows many of the important hallmarks of cancer angiogenesis found in vivo, including tip cells induction and migration and stalk cells formation. Importantly, the stalk cells develop a perfusable lumen that is connected to the parental vessel as demonstrated with perfusion of high-molecular-weight (150KD) fitc-dextran through microvascular structures. This model will be used as an in vitro cancer screening platform to unravel the important drivers in angiogenesis and vasculogenesis and the mechanism of action of antiangiogenic compounds. By combining this culture platform with mural cells, cell-cell interactions can be studied. In parallel, we will combine this 3D cancer angiogenesis platform with our current Tumor-on-a-Chip models to create tissue models with integrated vasculature. Citation Format: K M. Bircsak, V van Duinen, S J. Trietsch, A J. van Zonneveld, T Hankemeier, A Saleh, P Vulto. Perfusable 3D angiogenesis in a high-throughput microfluidic culture platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2051.

Because microvascular disease is one of the most important drivers of diabetic complications, early monitoring of microvascular integrity may be of clinical value. By assessing profiles of circulating microRNAs (miRNAs), known regulators of microvascular pathophysiology, in healthy controls and diabetic nephropathy (DN) patients before and after simultaneous pancreas-kidney transplantation (SPK), we aimed to identify differentially expressed miRNAs that associate with microvascular impairment. Following a pilot study, we selected 13 candidate miRNAs and determined their circulating levels in DN (n = 21), SPK-patients (n = 37), healthy controls (n = 19), type 1 diabetes mellitus patients (n = 15) and DN patients with a kidney transplant (n = 15). For validation of selected miRNAs, 14 DN patients were studied longitudinally up to 12 months after SPK. We demonstrated a direct association of miR-25, -27a, -126, -130b, -132, -152, -181a, -223, -320, -326, -340, -574-3p and -660 with DN. Of those, miR-25, -27a, -130b, -132, -152, -320, -326, -340, -574-3p and -660 normalized after SPK. Importantly, circulating levels of some of these miRNAs tightly associate with microvascular impairment as they relate to aberrant capillary tortuosity, angiopoietin-2/angiopoietin-1 ratios, circulating levels of soluble-thrombomodulin and insulin-like growth factor. Taken together, circulating miRNA profiles associate with DN and systemic microvascular damage, and might serve to identify individuals at risk of experiencing microvascular complications, as well as give insight into underlying pathologies.

Objectives Maturation failure is the major limitation of arteriovenous fistulas (AVFs) as hemodialysis access conduits. Indeed, 30–50% of AVFs fail to mature due to intimal hyperplasia and insufficient outward remodeling. Elastin has emerged as an important determinant of vascular remodeling. Here the role of elastin in AVF remodeling in elastin haplodeficient (eln +/– ) mice undergoing AVF surgery has been studied. Methods Unilateral AVFs between the branch of the jugular vein and carotid artery in an end to side manner were created in wild-type (WT) C57BL/6 ( n = 11) and in eln +/– mice ( n = 9). Animals were killed at day 21 and the AVFs were analyzed histologically and at an mRNA level using real-time quantitative polymerase chain reaction. Results Before AVF surgery, a marked reduction in elastin density in the internal elastic lamina (IEL) of eln +/– mice was observed. AVF surgery resulted in fragmentation of the venous internal elastic lamina in both groups while the expression of the tropoelastin mRNA was 53% lower in the eln +/– mice than in WT mice ( p .001). At 21 days after AVF surgery, the circumference of the venous outflow tract of the AVF was 21% larger in the eln +/– mice than in the WT mice ( p = .037), indicating enhanced outward remodeling in the eln +/– mice. No significant difference in intimal hyperplasia was observed. The venous lumen of the AVF in the eln +/– mice was 53% larger than in the WT mice, although this difference was not statistically significant (eln +/– , 350,116 ± 45,073 μm 2 ; WT, 229,405 ± 40,453 μm 2 ; p = .064). Conclusions In a murine model, elastin has an important role in vascular remodeling following AVF creation, in which a lower amount of elastin results in enhanced outward remodeling. Interventions targeting elastin degradation might be a viable option in order to improve AVF maturation.

Heart failure (HF) poses a heavy burden on patients, their families and society. The syndrome of HF comes in two types: with reduced ejection fraction (HFrEF) and preserved ejection fraction (HFpEF). The latter is on the increase and predominantly present in women, especially the older ones. There is an urgent need for mortality-reducing drugs in HFpEF, a disease affecting around 5 % of those aged 65 years and over. HFpEF develops in patients with risk factors and comorbidities such as obesity, hypertension, diabetes, COPD, but also preeclampsia. These conditions are likely to drive microvascular disease with involvement of the coronary microvasculature, which may eventually evolve into HFpEF. Currently, the diagnosis of HFPEF relies mainly on echocardiography. There are no biomarkers that can help diagnose female microvascular disease or facilitate the diagnosis of (early stages of) HFpEF. Recently a Dutch consortium was initiated, Queen of Hearts, with support from the Netherlands Heart Foundation, with the aim to discover and validate biomarkers for diastolic dysfunction and HFpEF in women. These biomarkers come from innovative blood-derived sources such as extracellular vesicles and circulating cells. Within the Queen of Hearts consortium, we will pursue female biomarkers that have the potential for further evolution in assays with point of care capabilities. As a spin-off, the consortium will gain knowledge on gender-specific pathology of HFpEF, possibly opening up novel treatment options.

Progenitor cells in the kidney: Biology and therapeutic perspectives. The stem cell may be viewed as an engineer who can read the blue print and become the building. The role of this fascinating cell in physiology and pathophysiology has recently attracted a great deal of interest. The archetype of stem cells is the zygote: one cell capable of endless proliferation and differentiation into all tissue types in the human body. Historically, the differentiation of embryonic stem cells is seen as an irreversible process with restricting possibilities for differentiation leading finally to a terminally differentiated cell type. Stem cells have also been described in the adult. They were first defined in tissues with a high cell turnover like skin and gut. Today, stem cells have also been shown in tissues with no or low regenerative potential and turnover, like the kidney. Traditionally, adult stem cells were thought to be restricted in their differentiative and regenerative potential to the tissues in which they reside. However, the stem cell concept is changing rapidly as evidence is mounting that adult stem cells not only reside locally in specific niches, but may also be recruited from the circulation to actively participate in the regeneration of various tissues. Furthermore, reverse differentiation has been demonstrated. This means that highly specialized cell types are able to dedifferentiate and engage in stem cell like activities. Moreover, transdifferentiation of mature cells into different cell types has been reported. This paper will review our current knowledge on renal stem cells and progenitor cells. Specifically, it will discuss the role of progenitor cells and transdifferentiation in renal repair and maintenance. Finally, the potential clinical implications of these findings will be discussed.

Activation and dysfunction of endothelial cells play a prominent role in patho-physiological processes such as atherosclerosis. We describe the identification by differential display of 106 cytokine-responsive gene fragments from endothelial cells, activated by monocyte conditioned medium or tumor necrosis factor-. A minority of the fragments (22/106) represent known genes involved in various processes, including leukocyte trafficking, vesicular transport, cell cycle control, apoptosis, and cellular protection against oxidative stress. Full-length cDNA clones were obtained for five novel transcripts that were induced or repressed more than 10-fold in vitro. These novel human cDNAs CA2_1, CG12_1, GG10_2, AG8_1, and GG2_1 encode inhibitor of apoptosis protein-1 (hIAP-1), homologues of apolipoprotein-L, mouse rabkinesin-6, rat stannin, and a novel 188 amino acid protein, respectively. Expression of 4 novel transcripts is shown by in situ hybridization on healthy and atherosclerotic vascular tissue, using monocyte chemotactic protein-1 as a marker for inflammation. CA2_1 (hIAP-1) and AG8_1 are expressed by endothelial cells and macrophage foam cells of the inflamed vascular wall. CG12_1 (apolipoprotein-L like) was specifically expressed in endothelial cells lining the normal and atherosclerotic iliac artery and aorta. These results substantiate the complex change in the gene expression pattern of vascular endothelial cells, which accompanies the inflammatory reaction of atherosclerotic lesions.

Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of 215 cDNA clones revealed the presence of a total of 28 different partial cDNAs encoding small GTP-binding proteins. Two sequences corresponded to novel isoforms of Rab2 and Rab9. In addition, human analogues of Rab4b, Rab7, Rab9, Rab14 and Rab15 were identified. Besides Rab proteins, members of other subfamilies were detected as well. As a first step towards elucidation of the function of the different small GTP-binding proteins identified we have isolated full length cDNA corresponding to Rab30 from a human endothelial cell cDNA library. In order to assess the subcellular localization of Rab30, we expressed epitope-tagged Rab30 cDNA in monkey kidney COS-1 cells. Immunoelectron-microscopy of transfected COS-1 cells indicated that Rab30 is associated with Golgi stacks.

Summary The low density lipoprotein receptor-related protein (LRP) is a large membrane glycoprotein that is a member of the low density lipoprotein (LDL) receptor family of endocytic receptors. In contrast to the restricted ligand specificity of the LDL receptor, LRP can bind and internalize a remarkable spectrum of structurally-unrelated classes of ligands suggesting a role for the receptor in diverse physiological and patho-physiological processes ranging from lipoprotein metabolism, cell growth and cell migration to atherosclerosis and Alzheimer's disease. In this review we will summarize the current insights in the biology of LRP and particularly focus on the recent progress in our understanding of the molecular mechanisms that enable LRP to interact specifically with such a multitude of different ligands.

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor gene family, mediates the cellular uptake of a diversity of ligands. A folding chaperone, the 39-kDa receptor-associated protein (RAP) that resides in the early compartments of the secretory pathway inhibits the binding of all ligands to the receptor and may serve to prevent premature binding of ligands to the receptor during the trafficking to the cell surface. To elucidate the molecular interactions that underlie the interplay between the receptor, RAP, and the ligands, we have analyzed and delineated the binding sites of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA).PAI-1 complexes, RAP, and the anti-LRP Fab fragment Fab A8. To that end, we have generated a series of soluble recombinant fragments spanning the second cluster of complement-type repeats (C3-C10) and the amino-terminal flanking epidermal growth factor repeat (E4) of LRP (E4-C10; amino acids 787-1165). All fragments were expressed by stably transfected baby hamster kidney cells and purified by affinity chromatography. A detailed study of ligand binding to the fragments using surface plasmon resonance revealed the presence of three distinct, Ca2+-dependent ligand binding sites in the cluster II domain (Cl-II) of LRP. t-PA.PAI-1 complexes as well as PAI-1 bind to a domain located in the amino-terminal portion of Cl-II, spanning repeats E4-C3-C7. Adjacent to this site and partially overlapping is a high affinity RAP-binding site located on repeats C5-C7. Fab A8, a pseudo-ligand of the receptor, binds to a third Ca2+-dependent binding site on repeats C8-C10 at the carboxyl-terminal end of Cl-II. Next, we studied the RAP-mediated inhibition of ligand binding to LRP and to Cl-II. As expected, we observed a strong inhibition of t-PA.PAI-1 complex and Fab A8 binding to LRP by RAP (IC50 congruent with 0.3 nM), whereas in the reverse experiment, competition of t-PA. PAI-1 complexes and Fab A8 for RAP binding to LRP could only be shown at high concentrations of competitors (>/=1 microM). Interestingly, even though the equilibrium dissociation constants for the binding of RAP to LRP and to Cl-II are similar, the binding of the ligands to Cl-II is only prevented by RAP at concentrations that are at least 2 orders of magnitude higher than those required for inhibition of ligand binding to LRP. Our results favor models that propose RAP-induced allosteric inhibition of ligand binding to LRP that may require LRP moieties that are located outside Cl-II of the receptor.

Objective: Atherosclerosis is associated with increased levels of plasma cytokines and expression of Toll-like receptors (TLRs). Yet, little is known about the potential use of TLR ligand induced cytokine release as a biomarker of coronary artery disease (CAD). In this study, we investigated whether TLR ligand induced cytokine release is associated with atherosclerotic disease severity and its predictive value for future cardiovascular events. Methods: Blood samples were obtained from 260 patients with stable angina and 15 healthy controls. Cytokine levels of TNFα, IL-8 and IL-6 were measured after 2h of whole blood stimulation with 10ng/ml lipopolysaccharide (LPS, TLR4 ligand) and P3C 500ng/ml (TLR2 ligand). In a subgroup, dose-response curves were created using additional LPS concentrations. Results: LPS induced whole blood release of TNFα and IL-6, but not IL-8, was significantly higher in patients compared to healthy controls. Among CAD patients, TLR responses did hardly differ when associated with the presence of traditional risk factors and atherosclerotic disease severity (number of diseased vessels and coronary stenosis degree). Patients with secondary events during follow-up showed a trend towards an increased TLR response. Furthermore, positive associations were found between CRP levels and TLR-induced TNFα (CRP2: 2364pg/ml) and IL-6 production (CRP2: 2250pg/ml). Conclusion: In conclusion, TLR-induced whole blood cytokine release in patients with stable angina indicates the presence of coronary atherosclerosis but does not reflect its severity.

We describe a phage-display-based method to identify epitopes or interaction sites on proteins. DNA encoding the protein of interest is partially degraded with DNase I to generate random fragments of 50–200 bp. These fragments are then cloned into a phagemid vector that has been modified to allow the expression of the random fragments and the construction of a (bacterio)phage-displayed random epitope library. Phages displaying functional epitopes can be selected from these libraries by affinity selection or panning. To test this method we have constructed a random-epitope library for human plasminogen-activator inhibitor 1 and used this library to map the epitope of a monoclonal antibody (mAb) directed against this protein. By alignment of the selected overlapping epitope-containing fragments, we were able to locate the epitope of the mAb on a stretch of 39 amino acids spanning from E128 to V166. The approach may also be applied to more complex systems than single-protein genes, such as viral genomes or complete cDNA libraries.

Eryhropoiesis-stimulating agents have demonstrated tissue-protective effects in experimental models of ischemia-reperfusion injury. PROTECT was a 12-month, randomized, double-blind, placebo-controlled, single center study with high-dose recombinant human erythropoietin-β (Epoetin) in 92 donation after cardiac death (DCD) kidney transplant recipients. Patients were randomized to receive an intravenous bolus of Epoetin (3.3 × 10(4) international unit (IU); n = 45) or placebo (saline 0.9% solution; n = 47) on 3 consecutive days, starting 3-4 h before the transplantation and 24 h and 48 h after reperfusion. The immunosuppressive regimen included an anti-CD25 antibody, steroids, mycophenolate mofetil and delayed introduction of cyclosporine. Primary end point was a composite of the incidence of primary nonfunction and delayed graft function, either defined by spontaneous functional recovery or need for dialysis in the first week. Secondary objectives included duration of delayed function, renal function and proteinuria up to 1 year and thrombotic adverse events. Results showed no differences in the incidence or duration of delayed graft function and/or primary nonfunction (Epoetin 77.8 vs. placebo 78.7%, p = 1.00). Epoetin treatment significantly increased the risk of thrombotic events at 1 month and 1 year (Epoetin 24.4% vs. placebo 6.4%, p = 0.02).

Simultaneous pancreas-kidney transplantation (SPK) is an advanced treatment option for type 1 diabetes mellitus (DM) patients with microvascular disease including nephropathy. Sidestreamdarkfield (SDF) imaging has emerged as a noninvasive tool to visualize the human microcirculation. This study assessed the effect of SPK in diabetic nephropathy (DN) patients on microvascular alterations using SDF and correlated this with markers for endothelial dysfunction. Microvascular morphology was visualized using SDF of the oral mucosa in DN (n = 26) and SPK patients (n = 38), healthy controls (n = 20), DM1 patients (n = 15, DM ≥ 40 mL/min) and DN patients with a kidney transplant (KTx, n = 15). Furthermore, 21 DN patients were studied longitudinally up to 12 months after SPK. Circulating levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and soluble thrombomodulin (sTM) were measured using ELISA. Capillary tortuosity in the DN (1.83 ± 0.42) and DM ≥ 40 mL/min (1.55 ± 0.1) group was increased and showed reversal after SPK (1.31 ± 0.3, p < 0.001), but not after KTx (1.64 ± 0.1). sTM levels were increased in DN patients and reduced in SPK and KTx recipients (p < 0.05), while the Ang-2/Ang-1 ratio was normalized after SPK and not after KTx alone (from 0.16 ± 0.04 to 0.08 ± 0.02, p < 0.05). Interestingly, in the longitudinal study, reversal of capillary tortuosity and decrease in Ang-2/Ang-1 ratio and sTM was observed within 12 months after SPK. SPK is effective in reversing the systemic microvascular structural abnormalities in DN patients in the first year after transplantation.

Asgeirsdottir SA, van Solingen C, Kurniati NF, Zwiers PJ, Heeringa P, van Meurs M, Satchell SC, Saleem MA, Mathieson PW, Banas B, Kamps JA, Rabelink TJ, van Zonneveld AJ, Molema G. MicroRNA-126 contributes to renal microvascular heterogeneity of VCAM-1 protein expression in acute inflammation. Am J Physiol Renal Physiol 302: F1630-F1639, 2012. First published March 14, 2012; doi: 10.1152/ajprenal.00400.2011.-Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-alpha treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.

Endothelial to mesenchymal transition (EndoMT) has been proposed to be involved in the loss of microvascular capillaries in the pathophysiology of fibrosis and organ failure. In EndoMT, endothelial cells (EC) undergo a mesenchymal transition associated with the loss of cell-cell contacts and the acquisition of a synthetic, contractile phenotype. Here, we sought to identify microRNAs (miRNAs) that could play a central role in regulating EndoMT. In a TGF-β dependent in vitro model for EndoMT, we identified miRNAs that were differentially expressed in normoxic and hypoxic conditions. These studies identified miR-155 to be significantly upregulated in EndoMT, an effect that was enhanced under hypoxia, which further augments EndoMT. Silencing of miR-155 directly increased RhoA expression and activity in endothelial cells and affected phosphorylation of downstream LIMK. In contrast, overexpression of miR-155 counteracted RhoA function. Using a selective Rho kinase inhibitor, we could partly suppress EndoMT, strengthening the notion that RhoA plays a central role in EndoMT. Forced overexpression of miR-155 completely suppressed EndoMT, as evidenced by the maintenance of EC characteristics and blocking the acquisition of a mesenchymal phenotype, as compared to control cells. Our data demonstrate that miRNA-155 functions as a negative regulator of RhoA signaling in TGF-β-induced endothelial to mesenchymal transition.

Objective— The presence of kinase-insert domain-containing receptor (KDR) on circulating CD34 + cells is assumed to be indicative for the potential of these cells to support vascular maintenance and repair. However, in bone marrow and in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood, less than 0.5% of CD34 + cells co-express KDR. Therefore, we studied whether CD34 + /KDR + cells are generated in the peripheral circulation. Methods and Results— Using an ex vivo flow model, we show that activated platelets enable CD34 + cells to home to sites of vascular injury and that upon immobilization, KDR is translocated from an endosomal compartment to the cell-surface within 15 minutes. In patients with diabetes mellitus type 2, the percentage of circulating CD34 + co-expressing KDR was significantly elevated compared to age-matched controls. When treated with aspirin, the patients showed a 49% reduction in the generation of CD34 + /KDR + cells, indicating that the level of circulating CD34 + /KDR + cells also relates to in vivo platelet activation. Conclusion— Circulating CD34 + /KDR + are not mobilized from bone marrow as a predestined endothelial progenitor cell population but are mostly generated from circulating multipotent CD34 + cells at sites of vascular injury. Therefore, the number of circulating CD34 + /KDR + cells may serve as a marker for vascular injury.

The importance of a specific variable region in different serine proteases for the interaction with plasminogen activator inhibitor 1 (PAI-1) is studied. To that end, we have constructed a thrombin substitution variant, thrombin-VR1, in which the entire variable region 1 (VR1) of the protease domain (Phe-34 to Leu-40) has been replaced by the corresponding sequence (Phe-294 to Phe-305) of tissue-type plasminogen activator. The substitution resulted in a 2000-fold increase of the second-order rate constant of inhibition by PAI-1 (k2 = 2.2 x 10(6) M-1 s-1) as compared to alpha-thrombin (k2 = 1.1 x 10(3) M-1 s-1). Inhibition of thrombin-VR1 by PAI-1 is mediated by the formation of SDS-stable, enzyme-inhibitor complexes. The substitution did not affect the rate constant of inhibition by antithrombin III, whereas clotting efficiency and the rate of inhibition by heparin cofactor II were decreased 3-fold. These results demonstrate the importance and specificity of the protease domain VR1 region for the interaction of PAI-1 with its target proteases.

These authors contributed equally to this work.Because microvascular disease is one of the mostimportant drivers of diabetic complications, earlymonitoringofmicrovascularintegritymaybeofclinicalvalue. By assessing profiles of circulating microRNAs(miRNAs), known regulators of microvascular patho-physiology, in healthy controls anddiabetic nephropa-thy (DN) patients before and after simultaneouspancreas–kidney transplantation (SPK), we aimed toidentifydifferentiallyexpressedmiRNAsthatassociatewith microvascular impairment. Following a pilotstudy, we selected 13 candidate miRNAs and deter-mined their circulating levels in DN (n¼21), SPK-patients (n¼37), healthy controls (n¼19), type 1diabetes mellitus patients (n¼15) and DN patientswith a kidney transplant (n¼15). For validation ofselected miRNAs, 14 DN patients were studied longi-tudinallyup to12months afterSPK.Wedemonstrateda direct association of miR-25, -27a, -126, -130b, -132,-152, -181a, -223, -320, -326, -340, -574-3p and -660 withDN.Ofthose,miR-25,-27a,-130b,-132,-152,-320,-326,-340, -574-3p and -660 normalized after SPK. Impor-tantly, circulating levels of some of these miRNAstightlyassociatewithmicrovascularimpairmentastheyrelate to aberrant capillary tortuosity, angiopoietin-2/angiopoietin-1 ratios, circulating levels of soluble-thrombomodulin and insulin-like growth factor. Takentogether, circulating miRNA profiles associate withDN and systemic microvascular damage, and mightserve to identify individuals at risk of experiencingmicrovascularcomplications,aswellasgiveinsightintounderlying pathologies.Abbreviations:Ang-1,angiopoietin-1;Ang-2,angiopoie-tin-2; DM, diabetes mellitus; DN, diabetic nephropathy;IGF, insulin-like growth factor; KTx, kidney transplanta-tion; miRNA, microRNA; MTI, mean tortuosity index;SPK, simultaneous pancreas–kidney transplantation;SPKFU, simultaneous pancreas–kidney transplantationfollow-up; sTM, soluble thrombomodulinReceived 28 March 2014, revised 22 September 2014and accepted for publication 20 October 2014

Introduction: A third of patients with critical limb ischaemia (CLI) eventually require amputation. In spite of clinically successful revascularisation patients rarely return to their pre-morbid status, and often report no improvement in their functional outcomes, which may be due to an underlying musculopathy. Non-haematopoietic EPO-derivatives have been designed to retain only tissue-protective functions of EPO. We hypothesised that ARA-290 (EPO-derivative) may have tissue-protective potential that would represent a novel therapeutic adjunct in patients with CLI. Methods: The effect of EPO and ARA-290 in mediating cytoprotection was assessed firstly in vitro using skeletal myoblasts isolated from CLI and control donors, and a model of simulated ischaemia. Characterisation of CLI myoblasts was also performed, to assess their contractile, migratory and proliferative ability. Subsequently, an in vivo murine model of hindlimb ischaemia, which recapitulates the muscular pathology observed in CLI patients, was used to assess the potential of ARA-290 to improve functional, histological and perfusion outcomes. Results: Skeletal myoblasts were successfully isolated from CLI patients for the first time. CLI myoblasts and myotubes exhibited increased proliferative capacity but reduced migratory and contractile function and importantly a reduced susceptibility to a second ischaemic-insult compared with control myoblasts and myotubes. EPO and ARA-290 treatment led to significant improvements in myoblasts and myotube function and survival via the JAK2/ STAT3, PI3k/Akt and NFkB signalling pathways. In vivo, animals treated with EPO and ARA-290 demonstrated improved functional, histological and perfusion outcomes compared to vehicle-control treated animals. Conclusion: These studies demonstrate the potential of EPO and its derivatives to protect tissues and cells from ischaemic-injury and encourages the development of novel pharmacological therapies for use in patients with “no option” CLI or severe functional deficit.

The systemic pro-inflammatory state present in patients with rheumatoid arthritis (RA) accelerates the progression of atherosclerosis through chronic endothelial activation. Uncoupling of endothelial nitric oxide synthase plays a central role in the amplification of oxidative signalling pathways that chronically activate and, ultimately, injure the endothelium. Recent studies indicate that the resultant loss of endothelial integrity in patients with RA may also involve defects in the vascular regenerative potential provided by circulating endothelial progenitor cells (EPC). This is most likely the consequence of endothelial cell dysfunction in the bone marrow stroma, which hampers the mobilisation of these EPC to the circulation. In addition, mediators of systemic inflammation in RA can affect a second pathway of vascular regeneration. Under normal circumstances, myeloid CD14+ cells can adopt a pro-angiogenic phenotype that plays a key role in vascular remodelling and collateral formation. However, the chronic systemic inflammation observed in patients with RA may skew the differentiation of bone marrow and circulating CD14+ cells in such a way that these cells lose their capacity to support collateral formation, increasing the risk of cardiovascular disease. Taken together, in patients with RA, the impaired capacity of circulating cells to support vascular regeneration may comprise a novel pathway in the development of premature atherosclerosis.

BACKGROUND We investigated the effect of inhibition of plasminogen activator inhibitor-1 (PAI-1) activity by a murine monoclonal anti-human PAI-1 antibody (MAI-12) on in vitro thrombolysis and on in vivo thrombolysis and thrombus extension in an experimental animal model for thrombosis. METHODS AND RESULTS Thrombolysis, mediated by recombinant tissue-type plasminogen activator (rt-PA), was studied in an in vitro system consisting of fibrinogen, plasminogen, and thrombin. Addition of purified platelets during clot formation inhibited rt-PA-mediated thrombolysis in a dose-dependent manner. Platelet-dependent thrombolysis resistance could be completely neutralized by the monoclonal antibody MAI-12, supporting the notion that the observed resistance is due to PAI-1 released from alpha-granules of platelets. Subsequently, we monitored in vivo thrombolysis and thrombus extension of human whole blood thrombi that were allowed to form in rabbit jugular veins. During thrombus formation, either MAI-12 or an irrelevant antibody was incorporated. Thrombolysis and thrombus extension were simultaneously measured during endogenous thrombolysis or upon administration of different dosages of rt-PA. We observed that endogenous thrombolysis was enhanced in the presence of MAI-12 compared with the control antibody. Significantly, thrombus extension was partially prevented by MAI-12 and not by the control antibody irrespective of whether exogenous rt-PA was systematically administered. CONCLUSIONS These observations further confirm the essential role of PAI-1 in the regulation of the thrombolytic system and support the exploration of adjunctive therapy based on inhibition of PAI-1 activity in thrombolytic strategies.

Regulation of the human type 1 plasminogen activator inhibitor (PAI-1) promoter by transforming growth factor-beta (TGF beta) was studied. An 800-base pair fragment from the PAI-1 promoter and 5'-flanking region was fused to the firefly luciferase reporter gene and transfected into Hep3B human hepatoma cells. Treatment of the cells with TGF beta induced luciferase activity by more than 50-fold. Transfection studies using constructs with 5' or 3' deletions through this region revealed that two sequences were important in the TGF beta response. The first sequence was located in the proximal promoter (-49 to -87) and mediated an 11-fold induction with TGF beta, while the second more distal region (-636 to -740) contained two sequences which together mediated a 50-fold or greater response. Sequence comparison indicated that both of the responsive regions contained sequences with high homology to the AP-1 consensus binding site. Moreover, gel retardation analysis experiments demonstrated that both sequences bound a common nuclear protein, and that an oligonucleotide containing a consensus AP-1 sequence was able to compete for the binding of this common protein. Thus, the response of the PAI-1 gene to TGF beta is mediated by at least two separate regions, and both of these regions contain DNA sequences homologous to the AP-1 binding site.

The erythropoietin–erythropoietin-receptor (EPO–EPO-R) system has recently been identified as an important cellular survival pathway. Its presence has also been demonstrated in the kidney and identified as a therapeutic target to prevent loss of renal function. Part of the protective effects may be related to the action of erythropoietin on endothelial function and expansion of endothelial progenitor cells. This paper reviews current evidence for involvement of these mechanisms in EPO-mediated renoprotection.

Objective—Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization and are therefore of great interest for autologous cell therapies to treat ischemic vascular disease. However, the origin and functional properties of these EPCs are still in debate.Methods and Results—Here, ex vivo expanded murine EPCs were characterized in terms of phenotype, lineage potential, differentiation from bone marrow (BM) precursors, and their functional properties using endothelial NO synthase (eNOS)–green fluorescent protein transgenic mice. Despite high phenotypic overlap with macrophages and dendritic cells, EPCs displayed unique eNOS expression, endothelial lineage potential in colony assays, and angiogenic characteristics, but also immunologic properties such as interleukin-12p70 production and low levels of T-cell stimulation. The majority of EPCs developed from an immature, CD31+Ly6C+myeloid progenitor fraction in the BM. Addition of myeloid growth factors such as macrophage–colony-stimulating factor (M-CSF) and granulocyte/macrophage (GM)-CSF stimulated the expansion of spleen-derived EPCs but not BM-derived EPCs.Conclusion—The close relationship between EPCs and other myeloid lineages may add to the complexity of using them in cell therapy. Our mouse model could be a highly useful tool to characterize EPCs functionally and phenotypically, to explore the origin and optimize the isolation of EPC fractions for therapeutic neovascularization.

Objective— Bone marrow-derived progenitor cells play a role in vascular regeneration. However, their homing to areas of vascular injury is poorly understood. One of the earliest responses to an injury is the activation of coagulation and platelets. In this study we assessed the role of hemostatic components in the recruitment of CD34 + cells to sites of injury. Methods and Results— Using an ex vivo injury model, representing endothelial cell (EC) injury or vessel denudation, we studied homing of CD34 + under flow. Platelet aggregates facilitated initial tethering and rolling of CD34 + cells through interaction of P-selectin expressed by platelets and P-selectin glycoprotein ligand-1 (PSGL-1), expressed by CD34 + cells. Ligation of PSGL-1 activated adhesion molecules on CD34 + cells, ultimately leading to firm adhesion of CD34 + cells to tissue factor-expressing ECs or to fibrin-containing thrombi formed on subendothelium. We also demonstrate that fibrin-containing thrombi can support migration of CD34 + cells to the site of injury and subsequent differentiation toward a mature EC phenotype. Additionally, intravenously injected CD34 + cells homed in vivo to denuded arteries in the presence of endogenous leukocytes. Conclusions— We provide evidence that hemostatic factors, associated with vascular injury, provide a regulatory microenvironment for re-endothelialization mediated by circulating progenitor cells.

Objective— Restenosis remains a major late complication of percutaneous transluminal coronary angioplasty (PTCA), for which the development of prevention strategies has thus far been hampered by the lack of a representative and practical animal model. We have, therefore, developed a murine model of PTCA-induced restenosis. Methods and Results— Rigid probe angioplasty of pre-existing atherosclerotic lesions in the carotid arteries of ApoE-deficient mice was found to result in an increase in lesion size (0.14±0.04×10 5 μm 2 to 0.42±0.09×10 5 μm 2 , P =0.007) with a smooth muscle cell-rich, fibrotic lesion morphology. In an additional experiment, lesions were incubated immediately after angioplasty with adenovirus bearing an endothelial nitric oxide synthase (eNOS) transgene (Ad.APT.eNOS), or an “empty” control virus (Ad.APT.empty) at a titer of 1.5×10 9 pfu/mL. Ad.APT.eNOS treatment was seen to lead to a 73.1% reduction in plaque size (0.27±0.04×10 5 μm 2 versus 1.02±0.39×10 5 μm 2 , P =0.07), which translated to a significantly lowered average degree of stenosis (33.6±4.1% versus 74.6±14.0%, P =0.02). Ad.APT.eNOS also decreased lesional collagen content from 29.1% to 4.8% ( P Conclusion— We believe that we have established a representative murine model of postangioplasty restenosis, which may serve to elucidate the mechanisms underlying restenosis and to evaluate potential antirestenotic therapies.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated alpha(2)-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA.plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody alpha(2)MRalpha3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.

Activation of human, arterial endothelial cells (ECs) is an early event in the pathogenesis of atherosclerosis. To identify the repertoire of genes that are differentially expressed after activation, we used serial analysis of gene expression (SAGE) to compare the mRNA spectrum of quiescent ECs with that of ECs activated for 6h with a strong atherogenic stimulus. SAGE methodology generates concatenated 'tags' of 10bp that are derived from a specific mRNA. About 5% of over 12000 tags analyzed is derived from genes that are differentially expressed (at least 5-fold up- or downregulated). These transcript tags are derived from only 56 genes, close to 1% of the total number of analyzed genes. Among these 56 differentially expressed genes are 42 known genes, including the hallmark endothelial cell activation markers interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), plasminogen activator inhibitor 1 (PAI-1), Gro-alpha, Gro-beta and E-selectin. Differential transcription of a selection of the upregulated genes was confirmed by Northern blot analysis. A novel observation is the upregulation of activin betaA mRNA, a member of the transforming growth factor beta family. Apparent discrepancies between this novel technology and conventional methods are discussed. In conclusion, we demonstrate that for the application of SAGE, a moderate number of analyzed transcript tags suffices to reveal the significant alterations of EC transcription that results from a strong atherogenic stimulus.

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K(d) approximately 60 nM). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC(50) approximately 1 nM). In addition, a 12-fold molar excess of the physiological carrier of FVIII, i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of (125)I-labeled FVIII by LRP-expressing cells ( approximately 8 fmol/10(5) cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k(on) approximately 5 x 10(4) M(-1) s(-1); k(off) approximately 2.5 x 10(-3) s(-1); K(d) approximately 50 nM). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on the in vivo survival of FVIII.

Endothelium derived nitric oxide (NO) plays a major role in cardiovascular homeostasis. It has important anti-atherosclerotic properties which include regulation of vasomotor tone and vessel wall permeability, suppression of leucocyte adhesion to the endothelial surface, inhibition of platelet aggregation, and inhibition of vascular smooth muscle cell migration and proliferation. The important role of NO in the cardiovascular system is highlighted by key observations in animal models. Inhibition of endothelial NO synthase (eNOS), the enzyme that catalyses endothelial NO synthesis, accelerates atherogenesis. Similarly, genetic deletion of eNOS in mice leads to hypertension, defective vascular remodelling, vascular thrombosis, and enhanced leucocyte–endothelial cell interactions. In humans, all major cardiovascular risk factors, including hypercholesterolaemia, hypertension, diabetes, and smoking, have been associated with endothelial dysfunction, characterised by impaired NO bioavailability. Importantly, the impairment of NO mediated endothelial function is an independent predictor of adverse cardiac events.1 Taken together, current data strongly suggest that impaired NO activity is a crucial factor in the pathogenesis of cardiovascular disease. Improving endothelial NO bioavailability in vivo may reduce cardiovascular risk and has emerged as a major therapeutic goal. In vivo NO bioactivity is determined by the balance between synthesis and degradation of NO. The biosynthesis of endothelial NO is catalysed by the enzyme eNOS and requires the amino acid L-arginine, nicotinamide adenine dinucleotide phosphate (NADPH), and molecular oxygen as substrates, as well as several cofactors and prosthetic groups. Inactivation of NO may occur by its reaction with oxyhaemoglobin in erythrocytes but also by reacting with superoxide anions, resulting in the formation of peroxynitrite. …

We have recently observed that chitotriosidase, a chitinolytic enzyme, is secreted by activated human macrophages and is markedly elevated in plasma of Gaucher disease patients (Hollak, C. E. M., van Weely, S., van Oers, M. H. J., and Aerts, J. M. F. G. (1994) J. Clin. Invest. 93, 1288-1292). Here, we report on the cloning of the corresponding cDNA. The nucleotide sequence of the cloned cDNA predicts a protein with amino acid sequences identical to those established for purified chitotriosidase. Secretion of active chitotriosidase was obtained after transient transfection of COS-1 cells with the cloned cDNA, confirming its identity as chitotriosidase cDNA. Chitotriosidase contains several regions with high homology to those present in chitinases from different species belonging to family 18 of glycosyl hydrolases. Northern blot analysis shows that expression of chitotriosidase mRNA occurs only at a late stage of differentiation of monocytes to activated macrophages in culture. Our results show that, in contrast to previous beliefs, human macrophages can synthesize a functional chitinase, a highly conserved enzyme with a strongly regulated expression. This enzyme may play a role in the degradation of chitin-containing pathogens and can be used as a marker for specific disease states.

SummaryThe present study was undertaken in order to determine the recognition site for tissue-type plasminogen activator-plasminogen activator inhibitor type 1 [t-PA-PAI-1] complexes in rat liver in vivo and in vitro. After intravenous injection into rats t-PA-PAI-1 complexes were rapidly removed from the plasma and the liver took up 80% of the injected dose. Within the liver parenchymal and endothelial liver cells contributed mainly to the uptake of t-PA-PAI-1, and were responsible for 62% and 24% of the liver uptake, respectively. The interaction of t-PA- PAI-1 with isolated rat parenchymal liver cells was of high affinity (Kd 17 nM). A well-known antagonist of the α2-macroglobulin receptor (α2MR/low-density lipoprotein receptor-related protein (LRP), GST-39kDa protein (GST-39kDaP) efficiently inhibited the binding (IC50 0.7 nM) of t-PA-PAI-1 to rat parenchymal liver cells. The interaction of t-PA-PAI-1 with LRP on rat parenchymal liver cells was not Ca2+-dependent and is most probably mediated by a specific determinant on PAI-1, since an anti-PAI-1 monoclonal antibody inhibited the binding of t-PA-PAI-1, where as free t-PA did not. The binding of t-PA-PAI-1 to rat hepatocytes could not be inhibited by a complex of plasmin and α2-antiplasmin nor by various other ligands of LRP like β-VLDL and lactoferrin. Binding of t-PA-PAI-1 to rat parenchymal liver cells was followed by internalization and subsequent degradation in the lysosomal compartment.It is concluded that parenchymal and endothelial liver cells mediate the removal of t-PA-PAI-1 complexes from the circulation. LRP on rat parenchymal liver cells is responsible for the uptake and degradation of t-PA-PAI-1 and may therefore be important for the regulation of the t-PA levels in the circulation.

Gene transfer therapy in vascular surgery is on the horizon and will include the insertion of genes for anti-clotting proteins into the endothelial lining of vascular grafts and for genes controlling the proliferation of smooth muscle cells after endovascular intervention. Here we address the possibility of targeting genes to specific vascular cells using non-infectious methods, DEAEdextran or lipofectin complexes of reporter genes, to aid transfection of endothelial cells, smooth muscle cells and fibroblasts cultured from human umbilical veins or arteries. For these studies we used the firefly luciferase gene under control of several different promoters including those for the Rous sarcoma virus (RSV) and for tissue plasminogen activator type 1 (PAI-1). DEAEdextran mediated transfections resulted in low level, transient (2–5 days) expression of RSV-luciferase in all three cell types. Lipofectin mediated transfections resulted in a four-to-five-fold higher expression of RSV-luciferase in endothelial and smooth muscle cells, expression remaining fairly stable for up to 14 days. One particular PAI-1 promoter construct of 800 by was only half as effective as the RSV promoter in the expression of luciferase from smooth muscle cells, 82 ± 9 and 35 ± 11 ng mg −1 respectively ( p −1 respectively. These experiments demonstrate the possibilities of augmenting cultured vascular cells with foreign genes using lipofectin, a cationic lipid, for insertions into endothelial and smooth muscle cells. The expression of genes in specific cells can be achieved by varying the gene promoter, with an 800 by PAI-1 promoter being relatively specific for endothelial cells. Application of similar techniques could find future use to improve the patency of vascular grafts and reduce rates of restenosis after angioplasty.

Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, we have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5' flanking region of the gene revealed a perfect "TATA box" at position -28 to position -23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5' flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk- fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides -305 and +75 of the plasminogen activator inhibitor type 1 gene.

Heparin has been shown recently to stimulate the activity of human tissue-type plasminogen activator (t-PA). To investigate this effect further, mutant proteins lacking various domains of t-PA were screened for the ability to be stimulated by heparin. Those mutants harboring either the finger domain or the 2nd kringle were found to have enhanced enzymatic activity in the presence of heparin. Only mutants containing these structures would bind to heparin-agarose beads; monoclonal antibodies directed against these domains blocked binding. The stimulatory effect of heparin was more pronounced in finger-containing mutants than kringle-2 proteins. Earlier results had localized the fibrin-binding domains to the same two structures. Unlike heparin, the 2nd kringle was shown to be more important than the finger for fibrin stimulation. Our results have implications for producing recombinant t-PA variants for use in thrombolytic therapy.