Leonard P. Adam

Detection of Osteopontin in Calcified Human Aortic Valves

Cardiac valve calcification often results in obstruction of blood flow, which eventually leads to valve replacement. The molecular mechanisms resulting in valve calcification are unknown. Collagen and specific bone matrix proteins are thought to provide the framework for ectopic tissue calcification. This investigation was performed to determine whether the bone matrix protein osteopontin was present in calcified human aortic valves. Proteins extracted from human aortic valve tissue were subjected to polyacrylamide gel electrophoresis followed by Western blotting, using polyclonal antibodies directed against osteopontin. Fresh frozen tissue sections were also screened for osteopontin and macrophages using immunohistochemical techniques. Osteopontin was present in both heavily and minimally calcified aortic valves and absent in noncalcified purely regurgitant or normal aortic valves by both radioimmunoassay (n = 16) and immunohistochemical techniques (n = 8). Osteopontin colocalized with valvular calcific deposits, and macrophages were identified in the vicinity of osteopontin. These results, in addition to showing that osteopontin is present in calcified human aortic valves, suggest that osteopontin is a regulatory protein in pathological calcification. Identification of the cells producing osteopontin in abnormal cardiac valves and of proximate stimuli for its secretion may lead to novel therapeutic strategies to prevent and/or reverse calcific valve disease.

Activation of Mitogen-Activated Protein Kinase in Porcine Carotid Arteries

The thin-filament protein h-caldesmon (the high molecular weight isoform of caldesmon) is phosphorylated in resting and contracted porcine carotid arteries. Phosphorylation of h-caldesmon in intact tissue occurs at sites that are covalently modified by mitogen-activated protein kinase (MAPK) in vitro. In this study, we have evaluated MAPK activation in arteries in response to mechanical load and pharmacological stimulation. MAPK was extracted from resting and stimulated porcine carotid arteries and then partially purified by anion-exchange fast-performance liquid chromatography. MAPK activity was separated into two peaks corresponding to the tyrosine-phosphorylated 42- and 44-kD isoforms of MAPK (p42MAPK and p44MAPK, respectively). Of the total MAPK activity, 42% was associated with p42MAPK, and 58% was associated with p44MAPK, this percentage was not altered by stimulation of the muscles with either KCl (110 mmol/L) or phorbol 12,13-dibutyrate (PDBu, 1 mumol/L). Both p42MAPK and p44MAPK, purified from porcine carotid arteries, phosphorylated h-caldesmon at the same sites and to levels approaching or > 1 mol phosphate per mole protein. In unloaded muscle strips, MAPK activity was 39 pmol.min-1.mg protein-1 when assayed with the peptide substrate APRTPG-GRR. MAPK activity increased in response to incremental mechanical loading to a maximum of 99 pmol.min-1.mg protein-1 at 16 x 10(3) N/m2. MAPK activity could be further increased in loaded muscles by pharmacological stimulation. With KCl stimulation, MAPK activities rose to a peak of 205 pmol.min-1.mg protein-1 at 10 minutes and then declined to basal values at 30 and 60 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of mitogen‐activated protein kinase phosphorylation sequences in mammalian h‐Caldesmon

h‐Caldesmon in vascular smooth muscle is phosphorylated in response to pharmacologie stimulation. Although many kinases phosphorylate h‐caldesmon, in vitro, the responsible kinase in intact tissue is unknown. The sites of phosphorylation in caldesmon from intact canine aortas have recently been identified and are consensus sequences for a proline‐directed protein kinase. In this study, we investigated the phosphorylation of h‐caldesmon by mitogen‐activated protein kinase (MAPK). Purified, recombinant MAPK phosphorylated porcine stomach h‐caldesmon to a stoichiometry approaching 2 mol phosphate/mol protein. Phosphorylated h‐caldesmon was subjected to proteolysis and the phosphopeptides were purified by high performance liquid chromatography. Two major phosphopeptides were identified and sequenced. These two peptides, VTS ∗ PTKV and S ∗ PAPK, were identical to the sequences of the sites phosphorylated in intact tissue. Antibodies to several enzymes implicated in the cascade of activation of MAPK were used to evaluate vascular smooth muscle by Western blotting. All components were found to be present. These data suggest that MAPK can function as a ‘caldesmon kinase’ in vascular smooth muscle.

Activation of MAP Kinase In Vivo Follows Balloon Overstretch Injury of Porcine Coronary and Carotid Arteries

Vascular restenosis involves contraction, proliferation, and remodeling of the arterial wall in response to overstretch injury. Mitogen-activated protein kinases (MAPKs) are implicated in both contraction and proliferation of vascular smooth muscle (VSM), and studies of porcine carotid arterial muscle strips have shown that mechanical stretch leads to the activation of the extracellular signal-regulated kinase (ERK) family of MAPKs in vivo. We, therefore, analyzed the acute effect of mechanical overstretch injury on ERK-MAPK (herein referred to simply as MAPK) activity in porcine coronary and carotid arteries in vivo. Balloon angioplasty catheters were inflated to 6 atm three times over 5 minutes at a balloon-artery ratio of 1.2:1 in either porcine coronary or carotid arteries. The arteries were snap-frozen after angioplasty, and MAPK activity was measured. Angioplasty of the left anterior descending (LAD, n = 5), left circumflex (LCx, n = 5), and carotid (n = 5) arteries effected an increase in MAPK activity compared with the activity in uninstrumented right coronary arteries (RCAs) or carotid arteries from the same animals used for controls. Balloon angioplasty of carotid arteries led to an increase in MAPK activity that was 7.7-fold over the activity in control arteries and comparable to the activity in stretched carotid arterial muscle strips in vivo. The increase in coronary artery kinase activity on angioplasty was variable from animal to animal. The increase in MAPK activity over that in control arteries ranged from 4.5- to 31.7-fold (mean +/- SEM, 10.7 +/- 5.3) in the LAD and 1.8- to 31.3-fold (mean +/- SEM, 9.7 +/- 5.7) in the LCx. There were no apparent inherent differences in the levels of MAPK activity in the three different types of coronary arteries (RCA, LAD, and LCx) without instrumentation. MAPK activation occurs rapidly during angioplasty, suggesting that this kinase may play an early role in initiating the injury response in both porcine coronary and carotid arteries. MAPKs may be key enzymes targeted to treat or prevent restenosis.

Phosphorylation sequences in h-caldesmon from phorbol ester-stimulated canine aortas

The high molecular weight form of caldesmon (h‐caldesmon) is phosphorylated in vascular smooth muscle. The stoichiometry of caldesmon phosphorylation increases in response to stimulation of the muscle by several contractile agonists; however, the responsible kinase has not been identified. In this study, we have sequenced the phosphopeptides prepared from h‐caldesmon phosphorylated in vitro by protein kinase C (PKC) as well as the phosphopeptides prepared from caldesmon phosphorylated in intact canine aortas that were stimulated to contract with PDBu. PKC phosphorylated three sites located in the C terminus: GSS*LKIEE, AEFLNKS*VQK and NLWEKQS*VDK, while h‐caldesmon from intact tissue was phosphorylated at two separate sites also in the C terminus: VTS*PTKV and S*PAPK. By comparison to known substrate consensus sequences for various protein kinases these data suggest that h‐caldesmon is directly phosphorylated by a proline‐directed protein kinase and not by PKC.

Myosin light chain and caldesmon phosphorylation in arterial muscle stimulated with endothelin-1

Endothelin-1 contracts porcine carotid arterial smooth muscle with an ED50 of 10 nM. Contraction is associated with phosphorylation of the 20,000 dalton-regulatory light chain subunits of vascular myosin. Phosphopeptide mapping of light chains isolated from 32PO4-loaded muscle strips stimulated by endothelin-1 (5 x 10(-8) M) and comparison with maps generated from light chains phosphorylated in vitro or muscles stimulated with KCl (110 mM) or angiotensin-II (5 x 10(-8) M) indicates that Ca2(+)-calmodulin activation of myosin light chain kinase is a biochemical pathway stimulated by all three agonists. However, a small amount of phosphate (17%) was detected in a light chain peptide phosphorylated by protein kinase C. Endothelin-1 also stimulated phosphorylation of the thin filament protein, caldesmon, (from 0.35 mol PO4/mol caldesmon to 0.52 mol PO4/mol). Collectively, these results provide evidence that the effects of endothelin-1 on force generation and maintenance in vascular muscle may be dependent upon myosin light chain phosphorylation by Ca2+ calmodulin--requiring myosin light chain kinase and upon a thin filament mechanism that is modulated by phosphorylation of caldesmon.