Johannes Waltenberger

C-Reactive Protein in the Arterial Intima Role of C-Reactive Protein Receptor–Dependent Monocyte Recruitment in Atherogenesis

Infiltration of monocytes into the arterial wall is an early cellular event in atherogenesis. Recent evidence shows that C-reactive protein (CRP) is deposited in the arterial intima at sites of atherogenesis. In this study, we demonstrate that CRP deposition precedes the appearance of monocytes in early atherosclerotic lesions. CRP is chemotactic for freshly isolated human blood monocytes. A specific CRP receptor is demonstrated on monocytes in vitro as well as in vivo, and blockage of the receptor by use of a monoclonal anti-receptor antibody completely abolishes CRP-induced chemotaxis. CRP may play a major role in the recruitment of monocytes during atherogenesis.

Intracoronary infusion of mononuclear cells from bone marrow or peripheral blood compared with standard therapy in patients after acute myocardial infarction treated by primary percutaneous coronary intervention: results of the randomized controlled HEBE trial

AIMS Previous trials that investigated cell therapy as an adjunctive therapy after acute myocardial infarction (AMI) have shown conflicting results. We designed a randomized controlled trial to determine the effect of intracoronary infusion of mononuclear cells from bone marrow (BM) or peripheral blood in patients with AMI. METHODS AND RESULTS In a multicentre trial, 200 patients with large first AMI treated with primary percutaneous coronary intervention were randomly assigned to either intracoronary infusion of mononuclear BM cells (n = 69), mononuclear peripheral blood cells (n = 66), or standard therapy (without placebo infusion) (n = 65). Mononuclear cells were delivered intracoronary between 3 and 8 days after AMI. Regional and global left ventricular myocardial function and volumes were assessed by magnetic resonance imaging before randomization and at 4 months, and clinical events were reported. The primary endpoint of the percentage of dysfunctional left ventricular segments that improved during follow-up did not differ significantly between either of the treatment groups and control: 38.6 ± 24.7% in the BM group, 36.8 ± 20.9% in the peripheral blood group, and 42.4 ± 18.7% in the control group (P = 0.33 and P = 0.14). Improvement of left ventricular ejection fraction was 3.8 ± 7.4% in the BM group, 4.2 ± 6.2% in the peripheral blood group when compared with 4.0 ± 5.8% in the control group (P = 0.94 and P = 0.90). Furthermore, the three groups did not differ significantly in changes in left ventricular volumes, mass, and infarct size and had similar rates of clinical events. CONCLUSION Intracoronary infusion of mononuclear cells from BM or peripheral blood following AMI does not improve regional or global systolic myocardial function in the HEBE trial. REGISTRATION The Netherlands Trial Register #NTR166 (www.trialregister.nl) and the International Standard Randomised Controlled Trial, #ISRCTN95796863 (http://isrctn.org).

Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity

Platelet-derived endothelial cell growth factor (PD-ECGF), a protein which stimulates angiogenesis in vivo, is shown to have a 39.2% amino acid sequence similarity over a 439 amino acid region with the thymidine phosphorylase of Escherichia coli (E. coli). Using recombinant human PD-ECGF, we show that PD-ECGF has thymidine phosphorylase activity. Analysis by gel chromatography revealed that recombinant human PD-ECGF occurs as a 90 kDa homodimer, similar to other thymidine phosphorylases. In addition to a possible effect on DNA synthesis, PD-ECGF was shown to affect [3H]thymidine assays in a manner which is not related to cell proliferation. The in vitro and in vivo effects of PD-ECGF may thus occur by an indirect mechanism through its enzymatic activity.

A Dual Inhibitor of Platelet-Derived Growth Factor β-Receptor and Src Kinase Activity Potently Interferes With Motogenic and Mitogenic Responses to PDGF in Vascular Smooth Muscle Cells A Novel Candidate for Prevention of Vascular Remodeling

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

Expression of protein tyrosine kinases in islet cells : possible role of the Flk-1 receptor for beta-cell maturation from duct cells.

To elucidate the expression of genes of importance for beta-cell replication and the production of insulin, single-stranded cDNAs from different preparations of insulin producing cells were used as template for the polymerase chain reaction (PCR) using primers specific for protein tyrosine kinases (PTKs). In RINm5F cells, as well as in fetal rat islets, the receptor PTK fetal liver kinase-1 (Flk-1) was expressed among other receptor and cytoplasmic tyrosine kinases. To elucidate the putative effects of stimulation of the Flk-1 receptor, fetal rat islet-like structures were cultured in the presence of the ligand for this receptor, vascular endothelial growth factor (VEGF). VEGF was found to stimulate both the insulin content/islet DNA ratio and the accumulation of insulin in the culture medium without affecting the rates of beta-cell replication. To investigate the localization of expression of the Flk-1 receptor in the pancreas, serial sections of fetal pancreata were immunostained for Flk-1 and insulin. Expression of Flk-1 was detected in endothelial-like cells and cells lining pancreatic ducts. The latter are considered to contain precursor cells for the endocrine pancreas. In conclusion, specific protein tyrosine kinases are expressed in islet cells, and are presumably participating in the regulation of islet function. Specifically, the receptor PTK Flk-1 may play a role of beta-cell maturation from pancreatic duct cells.

Essential role of calcium in vascular endothelial growth factor A-induced signaling: mechanism of the antiangiogenic effect of carboxyamidotriazole.

Vascular endothelial growth factor‐α (VEGF‐A) plays a major role in tumor angiogenesis and raises the concentration of intracellular free calcium ([Ca2+]i). Carboxyamidotriazole (CAI), an inhibitor of calcium influx and of angiogenesis, is under investigation as a tumoristatic agent. We studied the effect of CAI and the role of [Ca2+]i in VEGF‐α signaling in human endothelial cells. VEGF‐α induced a biphasic [Ca2+]i signal. VEGF‐α increased the level of intracellular inositol 1,4,5‐trisphosphate (IP3), which suggests that VEGF‐A releases Ca2+ from IP3‐sensitive stores and induces store‐operated calcium influx. Reduction of either extracellular or intracellular free Ca2+ inhibited VEGF‐A‐induced proliferation. CAI inhibited IP3 formation, both phases of the calcium signal, nitric oxide (NO) release, and proliferation induced by VEGF‐A. CAI prevented neither activation of VEGF receptor‐2 (VEGFR‐2) (KDR/Flk‐1), phospholipase C‐γ, or mitogen‐activated protein kinase (MAP kinase) nor translocation of nuclear factor of activated T cells (NFAT). We conclude that calcium signaling is necessary for VEGF‐A‐induced proliferation. MAP kinase activation occurs independently of [Ca2+]i but is not sufficient to induce proliferation in the absence of calcium signaling. Inhibition of the VEGF‐Ainduced [Ca2+]i signal and proliferation by CAI can be explained by inhibition of IP3 formation and may contribute to the antiangiogenic action of CAI. Calcium‐dependent NO formation may represent a link between calcium signaling and proliferation.

Ischemia-Induced Transplant Arteriosclerosis in the Rat

The effect of cold graft ischemia time on the development of transplant arteriosclerosis was investigated. Aorta grafts from DA or PVG rats were stored in a cold perfusion solution for 1, 4, or 24 hours before being orthotopically transplanted to PVG recipients. After observation times ranging from 2 to 8 weeks, the grafts were examined for various cell populations. Regional changes in the intima and media layers were measured by using an image analysis system. The arteriosclerosis-like changes seen in syngeneic grafts with the longest ischemia time could be almost as prominent as those seen in the allogeneic transplants. The magnitude of the regional intima changes in the syngeneic group correlated well with the ischemia time and in the allogeneic group with the observation time. The cell composition found in the intima and media of the allogeneic vessels consisted of macrophages, T-lymphocytes, MHC class II-expressing cells, and smooth muscle cells, whereas the syngeneic grafts contained almost exclusively smooth muscle cells and macrophages. We therefore conclude that the damage due to prolonged cold ischemia time is sufficient to cause pronounced graft arteriosclerosis. The pathophysiological mechanism leading to ischemia-induced arteriosclerosis is different from the one seen in the allogeneic situation.

The Molecular Basis of VEGFR-1 Signal Transduction Pathways in Primary Human Monocytes

Objective—Arteriogenesis, the growth of preexisting arterioles into functional arteries, is dependent on the proper function of monocytes. Likewise, wound healing is monocyte-dependent. The activation of vascular endothelial growth factor receptor-1 (VEGFR-1) in monocytes induces a chemotactic response, triggers the expression of tissue factor, and gene expression of cytokines and chemokines. Little is known about intracellular signaling pathways mediating the biological functions triggered by VEGFR-1 in primary monocytes. Methods and Results—Monocytes were isolated from peripheral venous blood of young healthy individuals using indirect magnetic labeling. Stimulation of monocytes with either vascular endothelial growth factor-A (VEGF-A) or placenta growth factor (PlGF-1) triggered VEGFR-1 autophosphorylation and phosphorylation of distinct downstream proteins: phosphatidylinositol-3 kinase (PI-3K), Akt, p38, and extracellular signal–regulated kinase-1/2 (ERK1/2). PI-3K appears to be a central regulator in VEGFR-1 signaling in monocytes as the activation of Akt, p38, and ERK1/2 are PI-3-K–dependent. In addition, Akt activation functions downstream of p38 kinase. VEGFR-1–mediated chemotaxis of monocytes is dependent on the activation of PI-3K, p38 kinase, Akt, and ERK1/2, when assessed in a modified Boyden chamber. Conclusions—Both PlGF-1 and VEGF-A can activate VEGFR-1–dependent signaling pathways in primary human monocytes, leading to the activation of several intracellular signaling pathways. These pathways are critically involved in primary monocyte chemotaxis.

Chemically sulfated Escherichia coli K5 polysaccharide derivatives as extracellular HIV-1 Tat protein antagonists

The HIV‐1 transactivating factor (Tat) acts as an extracellular cytokine on target cells, including endothelium. Here, we report about the Tat‐antagonist capacity of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. O‐sulfated K5 with high sulfation degree (K5‐OS(H)) and N,O‐sulfated K5 with high (K5‐N,OS(H)) or low (K5‐N,OS(L)) sulfation degree, but not unmodified K5, N‐sulfated K5, and O‐sulfated K5 with low sulfation degree, bind to Tat preventing its interaction with cell surface heparan sulfate proteoglycans, cell internalization, and consequent HIV‐LTR‐transactivation. Also, K5‐OS(H) and K5‐N,OS(H) prevent the interaction of Tat to the vascular endothelial growth factor receptor‐2 on endothelial cell (EC) surface. Finally, K5‐OS(H) inhibits αvβ3 integrin/Tat interaction and EC adhesion to immobilized Tat. Consequently, K5‐OS(H) and K5‐N,OS(H) inhibit the angiogenic activity of Tat in vivo. In conclusion, K5 derivatives with distinct sulfation patterns bind extracellular Tat and modulate its interaction with cell surface receptors and affect its biological activities. These findings provide the basis for the design of novel extracellular Tat antagonists with possible implications in anti‐AIDS therapies.

Intracoronary infusion of autologous mononuclear bone marrow cells in patients with acute myocardial infarction treated with primary PCI: Pilot study of the multicenter HEBE trial

Objective: This study was a pilot trial to determine safety and feasibility of intracoronary infusion of mononuclear bone marrow cells (MBMC) in patients with acute myocardial infarction (MI). Background: Studies reporting the effect of MBMC therapy on improvement of left ventricular (LV) function have shown variable results. The HEBE trial is a large multicenter, randomized trial that currently enrolls patients. Prior to this trial we performed a pilot study. Methods: Twenty‐six patients with a first acute MI were prospectively enrolled in eight centers. Bone marrow aspiration was performed at a median of 6 days after primary PCI (interquartile range, 5–7 days). MBMC were isolated by gradient centrifugation and were infused intracoronary the same day. All patients underwent magnetic resonance imaging before cell infusion and after 4 months. Clinical events were assessed up to 12 months. Results: Within 10 hr after bone marrow aspiration, 246 ± 133 × 106 MBMC were infused, of which 3.9 ± 2.3 × 106 cells were CD34+. In one patient, this procedure was complicated by local dissection. LV ejection fraction significantly increased from 45.0 ± 6.3% to 47.2 ± 6.5% (P = 0.03). Systolic wall thickening in dysfunctional segments at baseline improved with 0.9 ± 0.7 mm (P < 0.001). Infarct size decreased 37% from 17.8 ± 8.2 to 11.2 ± 4.2 gram (P < 0.001). During 12‐month follow‐up, 3 additional revascularizations were performed and an ICD was implanted in one patient, 3 weeks after PCI. Conclusion: In patients with acute MI, intracoronary infusion of MBMC is safe in a multicenter setting. At 4‐month follow‐up, a modest increase in global and regional LV function was observed, with a concomitant decrease in infarct size.