A cylindrical segment, free of complex atherosclerotic lesions, was resected at autopsy from each of 59 descending human thoracic aortas by cutting just below the level of the first pair of intercostal arteries and 35 mm distal to this incision. Each isolated tunica media was defatted and subjected to successive treatment with EDTA-Tris, 5 M guanidine hydrochloride-Tris, 5 M guanidine hydrochloride-Tris-DTE, collagenase and either trypsin or hot alkali. After each extraction or digestion, the dimensions and weight of the segments were measured and the extracted materials were analyzed and quantitated. This allowed the total content of the various components of the tunica media to be assessed by both gravimetric and analytical means. An age-related rise was observed in the total content of the following components: proteins and glycoproteins soluble in chaotropic solvents (ranging from 24 mg/cm in the youngest samples to 46 mg/cm in the oldest) and collagen (38 mg/cm to 69 mg/cm). In contrast, the total content of elastin remained constant at 70 mg/cm at all ages, but its concentration decreased due to the rise in the concentration of the other tissue components as the tunica media thickened with age. It was also noted that with increasing age there was an accumulation of protein(s) which could not be solubilized by extraction with chaotropic agents or with collagenase, but which could be removed by treatment with either trypsin or hot alkali. Mechanical measurements conducted before and after trypsin digestion on samples previously subjected to purification with the first four agents used suggest that this accumulated protein(s) influenced the elastic response of the tissue to the applied stress by increasing the incremental modulus, the breaking stress, and the hysteresis. After the removal of this additional protein(s), the mechanical behavior of the elastin component was found to be identical in all samples, irrespective of age. It is therefore proposed that the morphological changes and the stiffening observed in the aging aortic wall are not due to degradation of its elastin network but to variations in the supramolecular organization of connective tissue components.

Age-related changes in composition and mechanical properties of the tunica media of the upper thoracic human aorta.

Objective

We recently reported that rheumatoid arthritis (RA) patients had increased intima-media thickness (IMT) of the common carotid artery (CCA). The present longitudinal study was performed to determine whether the change in arterial thickness was accelerated in RA patients and to determine which factor was important in the progression of arterial wall changes.



Methods

We studied 62 female RA patients with stable disease activity and 63 healthy female controls. IMT of the CCA was measured twice by high-resolution B-mode ultrasonography. The second examination was performed 18–36 months after the first, and changes were expressed as millimeters of increase per year. Baseline examinations included blood markers of inflammation and urinary calcium excretion (expressed as the calcium-to-creatinine ratio).



Results

RA patients showed a significantly greater increase in IMT of the CCA compared with controls. In univariate analyses of the RA patient data, the C-reactive protein (CRP) level correlated with the increase in CCA IMT. Other markers of inflammation (the erythrocyte sedimentation rate and white blood cell and platelet counts) also showed significant positive associations with the annual increase in CCA IMT in multiple regression models when adjusted for age, smoking status, blood pressure, and serum cholesterol level. The urinary calcium-to-creatinine ratio was also significantly associated with an increase in CCA IMT. Moreover, both the CRP level and the urinary calcium-to-creatinine ratio were significantly and independently associated with the increase in IMT of the CCA.



Conclusion

Patients with RA have a higher rate of increase in thickening of the arterial wall. Inflammation and calcium mobilization are factors closely associated with the accelerated arterial wall changes.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/art.11327

Inflammation and bone resorption as independent factors of accelerated arterial wall thickening in patients with rheumatoid arthritis.

Gamma-carboxyglutamic acid is an amino acid with a dicarboxylic acid side chain. This amino acid, with unique metal binding properties, confers metal binding character to the proteins into which it is incorporated. This amino acid has been discovered in blood coagulation proteins (prothrombin, Factor X, Factor IX, and Factor VII), plasma proteins of unknown function (Protein C, Protein S, and Protein Z), and proteins from calcified tissue (osteocalcin and bone-Gla protein). It has also been observed in renal calculi, atherosclerotic plaque, and the egg chorioallantoic membrane, among other tissues. Gamma-carboxyglutamic acid is synthesized by the post-translational modification of glutamic acid residues. This reaction, catalyzed by a hepatic carboxylase, requires reduced vitamin K, oxygen, and carbon dioxide. The function of γ- carboxyglutamic acid is uncertain. In prothrombin γ-carboxyglutamic acid residues bound to metal ions participate as an intramolecular non-covalent bridge to maintain protein conformation. Additionally, these amino acids participate in the calcium-dependent molecular assembly of proteins on membrane surfaces through intermolecular bridges involving γ-carboxyglutamic acid and metal ions.

Gamma-carboxyglutamic acid

Vitamin K dependent carboxylation of glutamate residues to gamma-carboxyglutamate was demonstrated in proteins of spleen and testes microsomes. The rate of carboxylation in spleen microsomes was 0.9% and testes 3% of that in liver microsomes per milligram of microsomal protein. For comparison the rates of carboxylation in lung and kidney microsomes were 17 and 8%, respectively, of the rate in liver microsomes. The high rate in liver microsomes may be due to a high carboxylase level as indicated by the high rate of peptide carboxylation in liver microsomes. Protein carboxylation in liver microsomes was linear for only 15 min while carboxylation in microsomes from extrahepatic tissue persisted much longer so that the total protein carboxylation in lung microsomes was 60%, kidney 18%, testes 12%, and spleen 9% of that occurring in liver microsomes. Protein carboxylation was higher in microsomes from extrahepatic tissues of rats fed a vitamin K deficient diet as compared to animals fed a vitamin K sufficient diet. Protein carboxylation in microsomes from extrahepatic tissues was greatly stimulated by manganese ions and was dependent upon the addition of dithioerythritol. NADH could partially replace the dithiol in spleen, testes, and lung, but NADH-dependent carboxylation was relatively low in kidney and liver microsomes. Dithiol-dependent carboxylation was completely blocked by 10 microM warfarin, but NADH-dependent carboxylation was only slightly inhibited by 100 microM warfarin. Menaquinone-3 was much more active than vitamin K1 in driving carboxylation. Solubilized microsomes catalyzed the carboxylation of glutamate residues to gamma-carboxyglutamate in a pentapeptide Phe-Leu-Glu-Glu-Leu. The rate of carboxylation in lung microsomes was 22%, testes 3.3%, kidney 1.9%, and spleen 1.6% of the rate in liver microsomes.

Vitamin K dependent carboxylation of glutamate residues to gamma-carboxyglutamate in microsomes from spleen and testes: comparison with liver, lung, and kidney.

The determination of the sites of possible regulation in the formation of an active enzyme protein was only possible after the overall pathway of active enzyme synthesis had been established. Regulation may be via the triplet code at the genetic transcription level, or at the translation level from messenger RNA to protein or at a post-translational level via chemical modification and/or polypeptide aggregation. The fact that the role of vitamin K in the formation of the normally pro-active forms of the coagulation proteins (II, VII, IX, and X) was post- translational was shown by Goswami and Munro (1) in 1962, and further confirmed by Hill, et al. (2), Babior (3), Lowenthal and Simmons (4), Ranhotra, et al. (5), and Shah and Suttie (6). In these studies total enzyme activity (prothrombin) recovery in vivo was obtained following administration of vitamin K to vitamin K deficient (7,8) rats which had received protein synthesis blocking agents. R. E. Olson, et al. (9–12) stated that vitamin K functioned in prothrombin synthesis at a genetic or translation level on the basis of no response to the vitamin after synthesis of protein was inhibited. This, however, became untenable with the finding of carboxylation as the vitamin K dependent step (13–16) in prothrombin ‘completion’.

Olga Vancikova

Papers

The presence of γ-carboxyglutamic acid-containing protein in atheromatous aortae