Hans-Peter Holthoff

Essential myosin light chain as a target for caspase-3 in failing myocardium.

Programmed cell death involves the activation of caspase proteases that can mediate the cleavage of vital cytoskeletal proteins. We have recently reported that, in failing cardiac myocytes, caspase-3 activation is associated with a reduction in contractile performance. In this study we used a modified yeast two-hybrid system to screen for caspase-3 interacting proteins of the cardiac cytoskeleton. We identified ventricular essential myosin light chain (vMLC1) as a target for caspase-3. By sequencing and site-directed mutagenesis, a noncanonical cleavage site for caspase-3 was mapped to the C-terminal DFVE135G motif. We demonstrated that vMLC1 cleavage in failing myocardium in vivo is associated with a morphological disruption of the organized vMLC1 staining of sarcomeres, and with a reduction in myocyte contractile performance. Adenoviral gene transfer of the caspase inhibitor p35 in vivo prevented caspase-3 activation and vMLC1 cleavage, with positive impact on contractility. These data suggest that direct cleavage of vMLC1 by activated caspase-3 may contribute to depression of myocyte function by altering cross-bridge interaction between myosin and actin molecules. Therefore, activation of apoptotic pathways in the heart may lead to contractile dysfunction before cell death.

Administration of the cyclic peptide COR-1 in humans (phase I study): ex vivo measurements of anti-β1-adrenergic receptor antibody neutralization and of immune parameters

A novel concept for the treatment of heart failure is the neutralization of antibodies against the β1‐adrenergic receptor (anti‐β1AR‐ab). In a rat model of autoimmune cardiomyopathy, the cyclic peptide COR‐1 (given i.v. once monthly) neutralized anti‐β1AR‐abs and prevented anti‐β1AR‐ab‐induced myocardial damage, and completely reverted cardiac dysfunction over 3–6 months.

Effect of the oxLDL Binding Protein Fc-CD68 on Plaque Extension and Vulnerability in Atherosclerosis

Rationale: There is strong evidence that oxidative modification of low-density lipoprotein (oxLDL) plays a critical role in atherogenesis and that oxLDL may profoundly influence the mechanical stability of atherosclerotic plaques. Objective: To block oxLDL, we designed, expressed, and tested Fc-CD68, a soluble oxLDL binding protein consisting of human Fc and the extracellular domain of the human oxLDL-binding receptor CD68. Methods and Results: Fc-CD68 bound with high specific affinity to oxLDL and strongly bound and colocalized with oxLDL in plaques. To study the effects of repeated administrations of Fc-CD68 on the progression of atherosclerosis and plaque vulnerability, 12- and 16-week old cholesterol-fed ApoE−/− mice received either Fc-CD68 (n=6) or Fc control protein (n=6 to 8) thrice weekly for 4 weeks. Macroscopic and histological analysis of Sudan red lipid staining showed strong and significant reduction of plaque extension in the aorta and in the aortic root, respectively. Histological analysis of pentachrome- and Sirius-stained sections of the brachiocephalic arteries of 20 week-old ApoE−/− mice revealed that Fc-CD68 significantly reduced the occurrence of spontaneous ruptures of established plaques by ≈20%, compared with Fc and drastically increased the collagen content of plaques. Furthermore, in immunostained sections of the brachiocephalic artery and the aortic root, Fc-CD68 reduced the infiltration of plaques with T lymphocytes, and macrophages by ≈50% and 30%, respectively. Conclusions: The oxLDL binding protein Fc-CD68 attenuates atherosclerosis and strengthens the stability of atherosclerotic plaques.

The GPVI-Fc Fusion Protein Revacept Improves Cerebral Infarct Volume and Functional Outcome in Stroke

Objectives We examined the effect of Revacept, an Fc fusion protein which is specifically linked to the extracellular domain of glycoprotein VI (GPVI), on thrombus formation after vessel wall injury and on experimental stroke in mice. Background Several antiplatelet drugs for the treatment of myocardial infarction or ischemic stroke with potent anti-ischemic effects have been developed, but all incur a significant risk of bleeding. Methods Platelet adhesion and thrombus formation after endothelial injury was monitored in the carotid artery by intra-vital fluorescence microscopy. The morphological and clinical consequences of stroke were investigated in a mouse model with a one hour-occlusion of the middle cerebral artery. Results Thrombus formation was significantly decreased after endothelial injury by 1 mg/kg Revacept IV, compared to Fc only. 1 mg/kg Revacept IV applied in mice with ischemic stroke immediately before reperfusion significantly improved functional outcome, cerebral infarct size and edema compared to Fc only. Also treatment with 10 mg/kg rtPA was effective, and functional outcome was similar in both treatment groups. The combination of Revacept with rtPA leads to increased reperfusion compared to treatment with either agent alone. In contrast to rtPA, however, there were no signs of increased intracranial bleeding with Revacept. Both rtPA and Revacept improved survival after stroke compared to placebo treatment. Revacept and vWF bind to collagen and Revacept competitively prevented the binding of vWF to collagen. Conclusions Revacept reduces arterial thrombus formation, reduces cerebral infarct size and edema after ischemic stroke, improves functional and prognostic outcome without intracranial bleeding. Revacept not only prevents GPVI-mediated, but probably also vWF-mediated platelet adhesion and aggregate formation. Therefore Revacept might be a potent and safe tool to treat ischemic complications of stroke.

The GPVI-Fc fusion protein Revacept reduces thrombus formation and improves vascular dysfunction in atherosclerosis without any impact on bleeding times.

Aims Glycoprotein VI (GPVI) is a key platelet receptor which mediates plaque-induced platelet activation and consecutive atherothrombosis, but GPVI is also involved in platelet-mediated atheroprogression. Therefore, interference in GPVI-mediated platelet activation has the potential to combine short-term and long-term beneficial effects, specificity and safety especially regarding bleeding complications. Methods and Results We investigated the effects of the soluble dimeric GPVI receptor fusion protein, Revacept, an antagonist of collagen-mediated platelet activation, in an animal model of atherosclerosis: twenty week old rabbits, which had been fed on a cholesterol-rich diet for 8 weeks, received Revacept (8 mg/kg) or control twice weekly for 4 weeks. Pharmacokinetics indicated a slight accumulation of the drug in the serum after repeated dosing of Revacept for 3 weeks. A significant improvement of endothelial dysfunction after 0.06 and 0.6 µg/min acetylcholine and a significant decrease of vessel wall thickening were found after Revacept treatment. Accordingly, aortic vessel weight was reduced, and plaque sizes, macrophage and T-cell invasion tended to be reduced in histological evaluations. Bleeding time was determined after tail clipping in mice. Revacept alone or in combination with widely used anti-platelet drugs revealed a high safety margin with no prolongation of bleeding times. Conclusion Repeated doses of Revacept led to a significant improvement of endothelial dysfunction and vascular morphology in atherosclerotic rabbits. Furthermore, no influence of Revacept on bleeding time alone or in combinations with various anti-platelet drugs was found in mice. Thus, the inhibition of collagen-mediated platelet interaction with the atherosclerotic endothelium by Revacept exerts beneficial effects on morphology and vascular function in vivo and seems to have a wide therapeutic window without influencing the bleeding time.

Detection of Anti–β1-AR Autoantibodies in Heart Failure by a Cell-Based Competition ELISA

Rationale: Autoantibodies directed against the second extracellular loop of the cardiac &bgr;1-adrenergic receptor (&bgr;1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas heart disease. Various approaches have been used to detect such autoantibodies; however, the reported prevalence varies largely, depending on the detection method used. Objective: We analyzed sera from 167 DCM patients (ejection fraction <45%) and from 110 age-matched volunteers who did not report any heart disease themselves, with an often used simple peptide-ELISA approach, and compared it with a novel whole cell–based ELISA, using cells expressing the full transgene for the human &bgr;1-AR. Additionally, 35 patients with hypertensive heart disease with preserved ejection fraction were investigated. Methods and Results: The novel assay was designed according to the currently most reliable anti-TSH receptor antibody-ELISA used to diagnose Graves disease (“third-generation assay”) and also detects the target antibodies by competition with a specific monoclonal anti–&bgr;1-AR antibody (&bgr;1-AR MAb) directed against the functionally relevant &bgr;1-AR epitope. Anti–&bgr;1-AR antibodies were detected in ≈60% of DCM patients and in ≈8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti–&bgr;1-AR antibody titers (defined as inhibition of &bgr;1-AR MAb-binding) were no longer detected after depleting sera from IgG antibodies by protein G adsorption. In contrast, a previously used ELISA conducted with a linear 26-meric peptide derived from the second extracellular &bgr;1-AR loop yielded a high number of false-positive results precluding any specific identification of DCM patients. Conclusions: We established a simple and efficient screening assay detecting disease-relevant &bgr;1-AR autoantibodies in patient sera yielding a high reproducibility also in high throughput screening. The assay was validated according to “good laboratory practice” and can serve as a companion biodiagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure.

Structure Based Drug Design: Development of Potent and Selective Factor IXa (FIXa) Inhibitors

On the basis of our understanding on the binding interactions of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR strategy by targeting the 4-position of the template to access the S1 beta and S2-S4 sites. A number of highly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of functional groups with conformational changes toward the S2-S4 sites.

Overexpression of ABCG1 protein attenuates arteriosclerosis and endothelial dysfunction in atherosclerotic rabbits

The ABCG1 protein is centrally involved in reverse cholesterol transport from the vessel wall. Investigation of the effects of ABCG1 overexpression or knockdown in vivo has produced controversial results and strongly depended on the gene intervention model in which it was studied. Therefore, we investigated the effect of local overexpression of human ABCG1 in a novel model of vessel wall-directed adenoviral gene transfer in atherosclerotic rabbits. We conducted local, vascular-specific gene transfer by adenoviral delivery of human ABCG1 (Ad-ABCG1-GFP) in cholesterol-fed atherosclerotic rabbits in vivo. Endothelial overexpression of ABCG1 markedly reduced atheroprogression (plaque size) and almost blunted vascular inflammation, as shown by markedly reduced macrophage and smooth muscle cell invasion into the vascular wall. Also endothelial function, as determined by vascular ultrasound in vivo, was improved in rabbits after gene transfer with Ad-ABCG1-GFP. Therefore, both earlier and later stages of atherosclerosis were improved in this model of somatic gene transfer into the vessel wall. In contrast to results in transgenic mice, over-expression of ABCG1 by somatic gene transfer to the atherosclerotic vessel wall results in a significant improvement of plaque morphology and composition, and of vascular function in vivo.

Combined administration of the GPVI-Fc fusion protein Revacept with low-dose thrombolysis in the treatment of stroke

Background Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) remains the only approved medication for acute ischemic stroke, but incurs significant bleeding risks. Therefore, approaches to combine lower doses of thrombolytic therapy with other effective drugs aim at improving efficacy and reducing bleeding rates. We examined the safety and therapeutic effects of various dosings of rtPA, either alone or combined with glycoprotein VI-Fc fusion protein (GPVI-Fc, Revacept) on experimental stroke in mice. Methods and results The effect of filament-induced intracerebral thrombus formation and embolization was investigated after a one-hour occlusion of the middle cerebral artery. In accordance with previous studies, treatment with 10 mg/kg rtPA significantly improved functional outcome, cerebral infarct size and edema, but also resulted in markedly increased intracranial bleeding volumes. In contrast, low doses of rtPA (0.1 or 0.35 mg/kg body weight) did not change outcome parameters. However, addition of 1 mg/kg Revacept to 0.35 mg/kg rtPA led to improved reperfusion compared to rtPA alone. Moreover, these combined treatments resulted in improved grip strength, compared to the respective dose of rtPA alone. Infarct-surrounding edema improved after combined treatments, but not after respective single rtPA dosings. Intracranial bleeding volumes were below controls after all low-dose rtPA therapies, given either alone or combined with Revacept. Conclusions In contrast to using the equally effective full dose of rtPA, intracranial bleeding was not increased by low-dose rtPA combined with Revacept. Therefore, addition of Revacept to low-dose rtPA does not incur safety risks, but improves efficacy of treatment.

Cyclic Peptides for Effective Treatment in a Long-Term Model of Graves Disease and Orbitopathy in Female Mice

A model for human Graves disease in mice was used to compare several treatment approaches. The mice received regular adenovirus (Ad) thyroid-stimulating hormone receptor (TSHR) A subunit immunizations (injections every 4 weeks). The generation of anti-TSHR antibodies, enlarged thyroid sizes (goiter), elevated serum thyroxine levels, retro-orbital fibrosis, and cardiac involvement (tachycardia and hypertrophy) were consistently observed over 9 months. Treatment of established disease in these mice using cyclic peptides that mimic one of the cylindrical loops of the TSHR leucine-rich repeat domain improved or cured all investigated parameters after six consecutive monthly injections. The first significant beneficial effects were observed 3 to 4 months after starting these therapies. In immunologically naïve mice, administration of any of the cyclic peptides did not induce any immune response. In contrast, monthly injections of the full antigenic TSHR A domain as fusion protein with immunoglobulin G crystallizable fragment induced clinical signs of allergy in Ad-TSHR-immunized mice and anti-TSHR antibodies in naïve control mice. In conclusion, cyclic peptides resolved many clinical findings in a mouse model of established Graves disease and orbitopathy. In contrast to blocking TSHR by allosteric modulation, the approach does not incur a direct receptor antagonism, which might offer a favorable side effect profile.