Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

-AAB might have a mechanistic role and could represent a therapeutic target. in cardiomyocytes and induce mesentery artery segment contraction.-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension

A novel approach to induce cell cycle reentry in terminally differentiated muscle cells.

During terminal differentiation, skeletal muscle cells permanently retract from the cell cycle. We and others have shown previously that this cell cycle withdrawal is an actively maintained state that can be reversed by transient expression of the SV40 large T antigen. In an attempt to avoid the hazards of gene transfer and the difficulties of regulating transgene expression, we have now used this cellular system as a model to test whether direct protein delivery could constitute a feasible alternative or complementing strategy to gene therapy-based approaches. Taking advantage of the recently described intercellular trafficking properties of the herpes simplex virus I VP22 protein, we have constructed a chimeric VP22-SV40 large T antigen fusion protein and shown that it can spread into terminally differentiated myotubes where it accumulates in the nucleus. This fusion protein retains the ability to override the cell cycle arrest as shown for SV40 large T antigen alone. Our results clearly show that the transduced fusion protein remains capable of inducing S-phase and mitosis in these otherwise terminally differentiated cells and opens now the way to exploit this novel strategy for tissue regeneration

Effects of immunoadsorption on α₁-AAB and blood pressure.

<p>(A) Ordinate shows neonatal cardiomyocyte spontaneous beating rate; abscissa shows response to immunoadorption performed on a representative patient. The α₁-AAB activity decreased with every immunoadsorption with a total decrease in response over time. (B) Figure shows the initial spontaneous beating rate (closed circles). After five immunoadsorptions, this response is reduced to basal values (open circles) in all 5 patients. (C) Mean response of a representative patient in cardiomyocyte contraction assay over time to immunoadsorption is shown. (D) Mean arterial pressure (MAP) was measured 5 and 180 days after immunoadsorption. MAP was significantly reduced compared to before immunoadsorption.</p

Differential expression of <i>PLA2-IIA</i> and <i>Cacna1c</i> in cardiomyocytes and VSMC after treatment with patient α₁-AAB, rabbit rabbit α₁-AB or PE (Fold changes in TaqMan analysis).

<p>Differential expression of <i>PLA2-IIA</i> and <i>Cacna1c</i> in cardiomyocytes and VSMC after treatment with patient α₁-AAB, rabbit rabbit α₁-AB or PE (Fold changes in TaqMan analysis).</p

Epitope mapping for extracellular loop 1 and loop 2 of α_1Α-AR is given.

<p>(A) α₁-AAB were directed against the first extracellular loop (eL1) in 24% and against the second extracellular loop (eL2) in 27% of the hypertensive patients. (B) The peptide sequence P1 (YWAFGR) and P2 (GRVFCNI) (partially) for eL1 were able to diminish the spontaneous beating rate response completely. (C) Epitope mapping for loop 2 is given for the following amino acid sequences: P1: GWRQPA, P2: APEDET, P3: TICQIN, P4: INEEPG, P5: GYVLFS. The sequence P2 (APEDET) was able to diminish the spontaneous beating rate response completely.</p

Activation of ERK 1/2 in response to treatment with PE, α₁-AAB from patients or rabbit α₁-AB is shown.

<p>(A) Cardiomyocytes were treated for 5 and 15 min with 10 µM PE, 2.5 µg/ml α₁-AAB or rabbit α₁-AB, respectively. Equivalent amounts of protein were analyzed by Western blotting with anti-pERK 1/2 antibody (44/42 kD) and the amount of ERK was analyzed with anti-ERK 1/2 antibody. PE, patient α₁-AAB, and rabbit α₁-AB caused ERK 1/2 phosphorylation. (B) CHO/α_1Α-AR cells were incubated with patient α₁-AAB for 5 min. Lane 1 and 2 represent untreated cells. Patients 1, 4–8 were α₁-AAB-positive in the cardiomyocyte contraction assay. “Patient” 9 represents a pool of five α₁-AAB positive patients. Patients 10–12 were α₁-AAB negative in the cardiomyocyte contraction assay. Their serum samples were processed identically to those of α₁-AAB-positive patients. ERK 1/2 phosphorylation was inhibited by peptide P2, but not by peptide P5. Eukaryotic initiation factor 4E (elF4E, 25 kD) antibody was used as translational control, the amount of ERK was analyzed with anti-ERK 1/2 antibody.</p

Protein kinase C alpha (PKC-α) activation in cardiomyocytes and vascular smooth muscle cells (VSMC) after 2 min incubation with PE, α₁-AAB from patients, and rabbit α₁-AB is demonstrated.

<p>Horizontal rows show regimens, namely, untreated control, PE, patient α₁-AAB, and rabbit α₁-AB. Vertical columns show cardiomyocyte and VSMC responses. Yellow shows PKC-α activation; red is more intense activation. Gö 6976 is a PKC-α inhibitor, which blocked all responses.</p

Representative traces of responses in intracellular Ca²⁺ of cultivated neonatal rat cardiomyocytes exposed to isolated patient α₁-AAB and rabbit α₁-AB are shown.

<p>(A) α₁-AAB isolated from the serum of a patient with refractory hypertension elicited a Ca²⁺ signal. (B) The rabbit α₁-AB gave a similar Ca²⁺ signal. (C) A human control IgG preparation was unable to affect intracellular Ca²⁺. Cardiomyocytes were electrically stimulated at 1 Hz, and the peak Ca²⁺ was monitored.</p