The Ratio 1660/1690 cm−1 Measured by Infrared Microspectroscopy Is Not Specific of Enzymatic Collagen Cross-Links in Bone Tissue

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm−1 area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm−1 by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm−1 by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm−1 area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm−1 ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm−1 was unmodified. In conclusion, the 1660/1690 cm−1 is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process

Golgi, trafficking, and mitosis dysfunctions in pulmonary arterial endothelial cells exposed to monocrotaline pyrrole and NO scavenging

Although the administration of monocrotaline (MCT) into experimental animals is in widespread use today in investigations of pulmonary arterial hypertension (PAH), the underlying cellular and subcellular mechanisms that culminate in vascular remodeling are incompletely understood. Bovine pulmonary arterial endothelial cells (PAECs) in culture exposed to monocrotaline pyrrole (MCTP) develop “megalocytosis” 18–24 h later characterized by enlarged hyperploid cells with enlarged Golgi, mislocalization of endothelial nitric oxide synthase away from the plasma membrane, decreased cell-surface/caveolar nitric oxide (NO), and hypo-S-nitrosylation of caveolin-1, clathrin heavy chain, and N-ethylmaleimide-sensitive factor. We investigated whether MCTP did in fact affect functional intracellular trafficking. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) and the NO donor diethylamine NONOate were used for comparison. Both MCTP and c-PTIO produced distinctive four- to fivefold enlarged PAECs within 24–48 h with markedly enlarged/dispersed Golgi, as visualized by immunostaining for the Golgi tethers/matrix proteins giantin, GM130, and p115. Live-cell uptake of the Golgi marker C5 ceramide revealed a compact juxtanuclear Golgi in untreated PAECs, brightly labeled enlarged circumnuclear Golgi after MCTP, but minimally labeled Golgi elements after c-PTIO. These Golgi changes were reduced by NONOate. After an initial inhibition during the first day, both MCTP and c-PTIO markedly enhanced anterograde secretion of soluble cargo (exogenous vector-expressed recombinant horseradish peroxidase) over the next 4 days. Live-cell internalization assays using fluorescently tagged ligands showed that both MCTP and c-PTIO inhibited the retrograde uptake of acetylated low-density lipoprotein, transferrin, and cholera toxin B. Moreover, MCTP, and to a variable extent c-PTIO, reduced the cell-surface density of all receptors assayed (LDLR, TfnR, BMPR, Tie-2, and PECAM-1/CD31). In an important distinction, c-PTIO enhanced mitosis in PAECs but MCTP inhibited mitosis, even that due to c-PTIO, despite markedly exaggerated Golgi dispersal. Taken together, these data define a broad-spectrum Golgi and subcellular trafficking dysfunction syndrome in endothelial cells exposed to MCTP or NO scavenging