Tibetan medicine Duoxuekang ameliorates hypobaric hypoxia-induced brain injury in mice by restoration of cerebrovascular function.

ETHNOPHARMACOLOGICAL RELEVANCE: Duoxuekang (DXK, ཁྲག་འཕེལ་བདེ་བྱེད།) is a clinical experience prescription of CuoRu-Cailang, a famous Tibetan medicine master, which has effective advantages in the treatment of hypobaric hypoxia (HH)-induced brain injury. However, its underlying mechanisms remain unclear. AIM OF THE STUDY: The present study was designed to investigate the effects of DXK on cerebrovascular function of HH-induced brain injury in mice. MATERIALS AND METHODS: DSC-MR imaging was used to evaluate the effect of DXK on the brain blood perfusion of patients with hypoxic brain injury. HPLC analysis was used to detect the content of salidroside, gallic acid, tyrosol, corilagin, ellagic acid, isorhamnetin, quercetin and gingerol in DXK. The model of HH-induced brain injury in mice was established by an animal hypobaric and hypoxic chamber. The BABL/c mice were randomly divided into six groups: control group, model group, Hongjingtian oral liquid group (HOL, 3.3 ml/kg) and DXK groups (0.9, 1.8 and 3.6 g/kg). All mice (except the control group) were intragastrically administrated for a continuous 7 days and put into the animal hypobaric and hypoxic chamber after the last intragastric administration. Hematoxylin-eosin staining was employed to evaluate the pathological changes of brain tissue. Masson and Weigert stainings were used to detect the content of collagen fibers and elastic fibers of brain, respectively. Routine blood test and biochemical kits were used to analyze hematological parameters and oxidative stress indices. Immunofluorescence staining was applied to detect the protein levels of VEGF, CD31/vWF and α-SMA. RESULTS: The results of DSC-MR imaging confirmed that DXK can increased CBV in the left temporal lobe while decreased MTT in the right frontal lobe, right temporal lobe and right occipital lobe of the brain. DXK contains salidroside, gallic acid, tyrosol, corilagin, ellagic acid, isorhamnetin, quercetin and gingerol. Compared with the model group, DXK can ameliorate the atrophy and deformation, and increase the number of pyramidal neurons in hippocampal CA3 area and cortical neurocytes. Masson and Weigert stainings results revealed that DXK can significantly increase the content of collagen fibers and elastic fibers in brain. Routine blood test results demonstrated that DXK can dramatically decrease the levels of WBC, MCH and MCHC, while increase RBC, HGB, HCT, MCV and PLT in the blood samples. Biochemical results revealed that DXK can markedly increase SOD, CAT and GSH activities, while decrease MDA activity. Immunofluorescence revealed that DXK can notably increase the protein levels of VEGF, CD31/vWF and α-SMA. CONCLUSIONS: In conclusion, this study proved that DXK can ameliorate HH-induced brain injury by improving brain blood perfusion, increasing the number of collagen and elastic fibers and inhibiting oxidative stress injury. The underlying mechanisms may be involved in maintaining the integrity of cerebrovascular endothelial cells and vascular function. However, further in vivo and in vitro investigations are still needed to elucidate the mechanisms of DXK on regulating cerebral blood vessels.

Circadian rhythms in skin and other elastic tissues.

Circadian rhythms are daily oscillations that, in mammals, are driven by both a master clock, located in the brain, and peripheral clocks in cells and tissues. Approximately 10% of the transcriptome, including extracellular matrix components, is estimated to be under circadian control. Whilst it has been established that certain collagens and extracellular matrix proteases are diurnally regulated (for example in tendon, cartilage and intervertebral disc) the role played by circadian rhythms in mediating elastic fiber homeostasis is poorly understood. Skin, arteries and lungs are dynamic, resilient, elastic fiber-rich organs and tissues. In skin, circadian rhythms influence cell migration and proliferation, wound healing and susceptibility of the tissues to damage (from protease activity, oxidative stress and ultraviolet radiation). In the cardiovascular system, blood pressure and heart rate also follow age-dependent circadian rhythms whilst the lungs exhibit diurnal variations in immune response. In order to better understand these processes it will be necessary to characterise diurnal changes in extracellular matrix biology. In particular, given the sensitivity of peripheral clocks to external factors, the timed delivery of interventions (chronotherapy) has the potential to significantly improve the efficacy of treatments designed to repair and regenerate damaged cutaneous, vascular and pulmonary tissues.

Curcumin enhances the production of major structural components of elastic fibers, elastin, and fibrillin-1, in normal human fibroblast cells.

Curcumin is the major component of the yellow extract derived from the rhizome of the Curcuma longa, which is also a main bioactive polyphenol and has been generally used as a spice, food additive, and herbal medicine. In this presented study, we found that curcumin can enhance the production of major structural components of elastic fibers, elastin, and fibrillin-1, in normal human fibroblast cells via increasing ELN and FBN1 promoters' activities. With 2 µM curcumin treatment, the enhanced tropoelastin and fibrillin-1 protein amounts in Detroit 551 cells were approximately 134 and 130% of control, respectively. Therefore, our results demonstrated that curcumin may be used as a functional compound and applied to drugs, foods, and cosmetics in the future.

Phytoestrogen, tanshinone IIA diminishes collagen deposition and stimulates new elastogenesis in cultures of human cardiac fibroblasts.

It has been previously reported that oral or intra-peritoneal administration of tanshinone IIA can alleviate the ventricular hypertrophy and fibrosis that develops in rats after experimental cardiac infarction. Our present studies, performed on cultures of human cardiac fibroblasts, investigated the mechanism by which tanshinone IIA produces these beneficial effects. We found that treatment of cardiac fibroblasts with 0.1-10µM tanshinone IIA significantly inhibited their deposition of collagen I, while enhancing production of new elastic fibers. Moreover, both anti-collagenogenic and pro-elastogenic effects of tanshinone IIA occurred only after selective activation of the G protein-coupled estrogen receptor (GPER). This subsequently leads to initiation of the PKA/CREB phosphorylation pathway that inversely modulated transcription of collagen I and elastin genes. Interestingly, treatment of human cardiac fibroblasts with tanshinone IIA additionally up-regulated the production of the 67-kDa elastin binding protein, which facilitates tropoelastin secretion, and increased synthesis of lysyl oxidase, catalyzing cross-linkings of tropoelastin. Moreover, tanshinone IIA also caused up-regulation in the synthesis of collagenolytic MMP-1, but down-regulated levels of elastolytic MMP-2 and MMP-9. In summary, our data validate a novel mechanism in which tanshinone IIA, interacting with a non-classic estrogen receptor, maintains the proper balance between the net deposition of collagen and elastin, allowing for optimal durability and resiliency of the newly deposited matrix.

Polymerase delta interacting protein 2 sustains vascular structure and function.

OBJECTIVE: On the basis of previous evidence that polymerase delta interacting protein 2 (Poldip2) increases reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) activity in vascular smooth muscle cells, we hypothesized that in vivo knockdown of Poldip2 would inhibit reactive oxygen species production and alter vascular function. APPROACH AND RESULTS: Because homozygous Poldip2 deletion is lethal, Poldip2(+/-) mice were used. Poldip2 mRNA and protein levels were reduced by ≈50% in Poldip2(+/-) aorta, with no change in p22phox, Nox1, Nox2, and Nox4 mRNAs. NADPH oxidase activity was also inhibited in Poldip2(+/-) tissue. Isolated aortas from Poldip2(+/-) mice demonstrated impaired phenylephrine and potassium chloride-induced contractions, increased stiffness, and reduced compliance associated with disruption of elastic lamellae and excessive extracellular matrix deposition. Collagen I secretion was elevated in cultured vascular smooth muscle cells from Poldip2(+/-) mice and restored by H2O2 supplementation, suggesting that this novel function of Poldip2 is mediated by reactive oxygen species. Furthermore, Poldip2(+/-) mice were protected against aortic dilatation in a model of experimental aneurysm, an effect consistent with increased collagen secretion. CONCLUSIONS: Poldip2 knockdown reduces H2O2 production in vivo, leading to increases in extracellular matrix, greater vascular stiffness, and impaired agonist-mediated contraction. Thus, unaltered expression of Poldip2 is necessary for vascular integrity and function.

Vitamin E attenuates homocysteine and cholesterol induced damage in rat aorta.

BACKGROUND: The aim of this study was to investigate the effect of vitamin E on homocysteine and cholesterol-induced damage of rat aorta. METHODS: Wistar rats (all fed with a vitamin E poor diet) were divided into five groups. Control group was fed with the diet only, the second group received 1 mg kg(-1) day(-1) L-methionine in drinking water, the third group was fed with 2% cholesterol containing diet, the fourth group received L-methionine and cholesterol together, and the fifth group was fed with L-methionine and cholesterol and received intramuscular injections of vitamin E. After 4 weeks serum homocysteine, cholesterol and vitamin E levels were measured; aortas were removed; collagen and elastin and the major extracellular matrix components were evaluated microscopically as indicators of aortic degeneration. Aortic collagen content was measured by a colorimetric hydroxyproline assay. RESULTS: Four-week diet supplementation with methionine and cholesterol caused a twofold increase in serum homocysteine and 22% increase in serum cholesterol levels; endothelial damage and degenerative alterations in the aortic media were observed, as indicated by the dissociation of elastic fibers and accumulation of collagen. Vitamin E completely prevented the accumulation of collagen and largely prevented aorta damage as shown by the morphological data. CONCLUSION: The results indicate that, even moderate increases in homocysteine and cholesterol levels are sufficient to induce vascular degeneration that may be prevented by vitamin E supplementation.

Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation.

Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. The treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E(NO)) was collected. The lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E(NO) concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-ß, NF-κB, IFN-γ, and 8-iso-prostaglandin F2α contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. In conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.

Polypodium leucotomos decreases UV-induced epidermal cell proliferation and enhances p53 expression and plasma antioxidant capacity in hairless mice.

A single dose of ultraviolet radiation (UVR) induces significant changes in blood and skin of hairless mice. Oral administration of a hydrophilic extract of the fern Polypodium leucotomos (PL, 300 mg/kg during 5 days before UVR and for two additional days after irradiation) modulates some of the effects of UVR. Most significantly, PL administration reduced the number of proliferating cells by 13%, increased the number of p53(+) cells by 63%, enhanced the antioxidant plasma capacity (ORAC) by 30% and reinforced the network of dermal elastic fibres. Western blot analysis of skin antioxidant-related enzymes failed to demonstrate significant changes caused by PL. Thus, the beneficial effect of PL likely owes to its antioxidant and anti-ROS properties rather than its modulation of the expression of endogenous antioxidant systems. These data provide mechanistic clues for its efficacy as a systemic photoprotective agent with antioxidant and anti-photo-ageing properties.

Intravesical oxybutynin protects the vesical wall against functional and smooth muscle changes in rabbits with detrusor overactivity.

INTRODUCTION AND HYPOTHESIS: The aim of this study was to evaluate the protective effect of intravesical oxybutynin on the bladder wall of rabbits with detrusor overactivity and partial bladder outlet obstruction (PBOO). METHODS: Forty-five North Folk male rabbits were randomly distributed into GI, used as control (n = 15), GII-PBOO (n = 14), and GIII-PBOO + intravesical oxybutynin (n = 15). Connective tissue and elastic fibers were quantified as volumetric density on picrosirius red and Weigert's Fuchsin-Resorcin-stained sections, respectively. RESULTS: In T2, bladder weight was significantly higher in animals in GII and GIII. Smooth muscle bundle diameter was increased by 42% in GII compared with GI (p < 0.02).Elastic fibers were significantly higher in GII and GIII as compared with control group. Collagen concentration in GIII and GII was significantly lower than G1 (p < 0.025). CONCLUSION: Intravesical oxybutynin protected against structural and functional detrusor modifications of the partially obstructed bladder.

Extracts from Glycine max (soybean) induce elastin synthesis and inhibit elastase activity.

Elastic fibres are essential extracellular matrix components of the skin, contributing to its resilience and elasticity. In the course of skin ageing, elastin synthesis is reduced, and elastase activity is accelerated, resulting in skin sagging and reduced skin elasticity. Our studies show that non-denatured Glycine max (soybean) extracts induced elastin promoter activity, inhibited elastase activity and protected elastic fibres from degradation by exogenous elastases in vitro. Mouse and swine skins topically treated with soybean extracts showed enhanced elastic fibre network and increased desmosine content. Elastin expression was also augmented in human skin transplanted onto SCID mice in response to soy treatment. These data suggest that non-denatured soybean extracts may be used as skin care agents to reduce the signs of skin ageing.

Prevention of aortic elastic lamina defects by losartan in apolipoprotein(E)-deficient mouse.

1. In a previous study, we identified prevalent internal elastic lamina (IEL) defects in the aorta of hyperlipidaemic apolipoprotein E (ApoE)-deficient mice that are thought to provide a structural basis for the development of atherosclerosis and intimal thickening. In the present study, we examined the effects of losartan, an angiotensin AT1 receptor antagonist, on the development of IEL defects. 2. Male 18-week-old ApoE-deficient mice (maintained on a normal diet) were treated with losartan (3 or 30 mg/kg per day) for 10 weeks via the drinking water. The IEL defects were quantified histologically by measuring the continuity of the IEL within the inner curvature of the aortic arch. 3. In untreated animals, there was an age-dependent increase in IEL defects from 7.2 ± 2.1% at 18 weeks to 13.8 ± 4.0% at 28 weeks. Treatment with the high dose of losartan significantly prevented the development of IEL defects (4.7 ± 1.3% at 28 weeks; P < 0.05 vs untreated). This effect was independent of changes in blood pressure or plasma lipid levels. Using quantitative real-time polymerase chain reaction, we found that the effects of losartan were not associated with changes in levels of matrix metalloproteinase (MMP)-2 and MMP-9, tissue inhibitor of matrix metalloproteinase-1 or inflammatory markers in the aorta. 4. The results suggest that the renin-angiotensin system may contribute to the development of aortic IEL defects in a blood pressure-independent manner.

Bipolar fractional radiofrequency treatment induces neoelastogenesis and neocollagenesis.

BACKGROUND: We recently introduced Renesis, a novel minimally invasive radiofrequency (RF) device, for the treatment of human skin. The wound healing response post-fractional RF (FRF) treatment was examined in human subjects. STUDY DESIGN: The FRF system delivered RF energy directly within the dermis via 5 micro-needle electrode pairs. Tissue temperature was held at 72 degrees C for 4 seconds using an intelligent feedback system. The wound healing response was evaluated histologically and by RT-PCR up to 10 weeks post-RF treatment. Neoelastogenesis and the role of heat shock proteins (HSPs) were assessed by immunohistochemistry. RESULTS: FRF treatment generated a RF thermal zone (RFTZ) pattern in the reticular dermis that consisted of zones of denatured collagen separated by zones of spared dermis. RFTZs were observed through day 28 post-treatment but were replaced by new dermal tissue by 10 weeks. HSP72 expression rapidly diminished after day 2 while HSP47 expression increased progressively through 10 weeks. Reticular dermal volume, cellularity, hyaluronic acid, and elastin content increased. RT-PCR studies revealed an immediate increase in IL-1beta, TNF-alpha, and MMP-13 while MMP-1, HSP72, HSP47, and TGF-beta levels increased by 2 days. We also observed a marked induction of tropoelastin, fibrillin, as well as procollagens 1 and 3 by 28 days post-treatment. CONCLUSION: Our study revealed a vigorous wound healing response is initiated post-treatment, with progressive increase in inflammatory cell infiltration from day 2 through 10 weeks. An active dermal remodeling process driven by the collagen chaperone HSP47 led to complete replacement of RFTZs with new collagen by 10 weeks post-treatment. Furthermore, using both immunohistochemical and PCR studies, we successfully demonstrated for the first time evidence of profound neoelastogenesis following RF treatment of human skin. The combination of neoelastogenesis and neocollagenesis induced by treatment with the FRF system may provide a reliable treatment option for skin laxity and/or rhytids.

Acupoints [corrected] and meridians: a histochemical study.

The aim of this study was to elucidate the relationship between the structural specificities of acupoints and meridians as well as their clinical effects. We processed 356 specimens, 287 of which from 48 adult and 2 newborn cadavers and the remaining 69 from living patients; samples were taken at three different levels: (1) beneath acupoints; (2) between meridians; (3) at a distance from meridians. We performed seven different staining to show the distribution of collagen fibers, reticular fibers, mucopolysaccharides (MPS), connective tissue, nerve threads, and blood vessels in specimens obtained from different areas. We found that some structural and biochemical discrepancies associated with acupoints and meridians including: (1) mucopolysaccharides (MPS), in particular acid MPS; (2) collagen fibers; (3) nerve endings. We discussed these findings from an anatomo-clinical point of view.

The influence of low-level laser therapy on biomodulation of collagen and elastic fibers.

The study of low-level laser therapy upon extracellular matrix elements is important to understand the wound healing process under this agent. However, little is known about the interference of laser light in relation to collagen and elastic fibers. Cutaneous wounds were performed on the back of 72 Wistar rats and a Ga-Al-As low-level laser was punctually applied with different energy densities. The animals were killed after 24, 48, 72 hours and 5, 7 and 14 days. Tissues were stained with hematoxilin-eosin, sirius red fast green and orcein and then analyzed. It was observed that the treated group exhibited larger reduction of edema and inflammatory infiltrate. The treated animals presented a larger expression of collagen and elastic fibers, although without statistical significance (p > 0.05). Treatment with a dosage of 4 J/cm(2) exhibited more expressive results than that with 8 J/cm(2). In this study, the authors concluded that low-level laser therapy contributed to a larger expression of collagen and elastic fibers during the early phases of the wound healing process.

Changes in extracellular matrix macromolecules in human gingiva after treatment with drugs inducing gingival overgrowth.

It is generally agreed that gingival overgrowth results from an increase in the levels of gingival extracellular macromolecules infiltrated with various numbers of inflammatory cells. The relative amounts of extracellular matrix macromolecules observed in 12 cases of gingival hyperplasia associated with the use of cyclosporin, hydantoin or nifedipine were compared with those obtained in a control group on the basis of histological and immunohistochemical investigations. From tissue sections, the quantification was by computerized morphometric analysis on a BFM 186 microcomputer to which were implemented the transformations of mathematical morphology. The area fractions (AA%) occupied by total collagen, type III and type IV collagen, vessels, fibroblasts, fibronectin and elastic fibres were estimated and compared. The overall histological aspects of drug-induced gingival overgrowth were similar, but quantification of different extracellular matrix components showed differences. In the nifedipine and cyclosporin groups, the area occupied by fibroblasts were not significantly greater than in healthy gingiva and chronic gingivitis. The area occupied by collagen was significantly greater in the nifedipine group than in the other pathological groups. Fibronectin was also strongly expressed in the nifedipine group, and the elastic fibre network was preserved in this group.

Biopersistence of cerium in the human respiratory tract and ultrastructural findings.

For diagnostic purposes, mineralogical analysis was performed in bronchoalveolar lavage fluid and lung tissue from a 58-year-old patient previously exposed to asbestos and rare earth dusts. No significant retention of asbestos was demonstrated in lung tissue by light microscopy (asbestos bodies) or transmission electron microscopy analysis (uncoated fibers). Particles containing rare earth (cerium, lanthanum) and phosphorus were identified in alveolar macrophages in bronchoalveolar lavage fluid, and cerium-containing particles accounted for 70% of particles observed in the lung tissue. Ultrastructural analysis of lung tissue revealed the presence of particles containing cerium and phosphorus in interstitial macrophages and elastic fibers. These results suggest that rare earth is metabolized and should be considered as biopersistent in the human respiratory tract, since occupational inquiries revealed that exposure to cerium oxide abrasive powder had ceased at least 15 years earlier.

Elastolytic activity of human monocytes from synovial fluid and blood of patients with arthritis. Relations to levels of interleukin 6 and soluble interleukin 2 receptor.

Synovial fluid (SF) and blood from 24 patients with non-traumatic, sterile hydarthron were examined for monocyte elastolysis (MøE) and for levels of interleukin 6 (IL-6) and of soluble interleukin 2 receptor (sIL-2R). Six patients had osteoarthrosis (OA) and 18 patients had inflammatory hydarthron (IH), 10 of whom had rheumatoid arthritis (RA). Blood MøE was lower in OA than in IH, both measured as basal MøE activity and after in vitro stimulation with immune complexes and phorbol myristate acetate (PMA). SF MøE was higher than MøE in blood (p less than 0.01). This increase in SF MøE could be mimicked in vitro by prestimulation of blood Mø with low levels of IC. SF IL-6 and sIL-2R were also elevated (p less than 0.01). All three parameters correlated to the degree of joint inflammation evaluated by SF leucocyte level, complement activation, blood C Reactive Protein, and to the clinical evaluation of the joint. The increase in SF MøE, IL-6 and sIL-2R in patients with IH, points to a stimulation of Mø and lymphocytes in the joint.

The mineralization of elastic fibers and alterations of extracellular matrix in pseudoxanthoma elasticum. Ultrastructure, immunocytochemistry, and X-ray analysis.

Histologic paraffin sections of pseudoxanthoma elasticum (PXE)-involved skin of forearm and axilla were used for histochemistry and immunohistochemical and analytical electron microscopy to study the progressive mineralization in the dermis of patients with PXE. The von Kossa technique identified mineral deposits throughout the reticular PXE dermis. X-ray analysis revealed patterns of calcium and phosphorus deposition in the von Kossa-positive areas, and the immunohistochemical staining using monoclonal antibodies identified increased chondroitin-6-sulfate in these areas when compared with normal skin. Scanning transmission electron microscopy observation combined with X-ray dot mapping show calcium and phosphorus to be codistributed within the mineralized area. This study confirms by new methods the increase in chondroitin-6-sulfate, alterations in elastin and collagen, and a high calcium and phosphorus elemental distribution matching the mineralized area in the PXE dermis.

The nature of the microfibrillar glycoproteins of elastic fibres. A biosynthetic study.

1. Cell cultures propagated from foetal bovine ligamentum nuchae synthesized and secreted two glycoproteins, designated MFP I and MFP II, that are closely related to elastic-fibre microfibrils. Glycoproteins MFP I (apparent mol.wt. 150 000) and MFP II (apparent mol.wt. 300 000) were metabolically labelled, separated from other culture-medium components by immunoprecipitation with a specific anti-(microfibrillar protein) serum, and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and sodium dodecyl sulphate/gel-filtration chromatography. 2. Ligament cells also synthesized and secreted fibronectin, but salt-fractionation and immunoprecipitation studies with a specific anti-(cold-insoluble globulin) serum established that neither glycoprotein MFP I nor glycoprotein MFP II was related to fibronectin. 3. The secretion of glycoprotein MFP I, but not that of glycoprotein MFP II, was enhanced by the addition of ascorbate to the culture medium. 4. Ascorbate-supplemented ligament cells incorporated [3H]proline into glycoprotein MFP I, and 36% of the nondiffusible proline residues were hydroxylated, exclusively as 4-hydroxy[3H]proline. Less than 1% of the total proline residues in [3H]proline-labelled glycoprotein MFP II were hydroxylated. 5. Ascorbate-supplemented cells incorporated [14C]lysine into glycoprotein MFP I and 30% of the non-diffusible lysine residues were hydroxylated. 6. Newly secreted glycoprotein MFP I was digested by highly purified bacterial collagenase to yield polypeptide fragments of apparent mol.wts. 50 000 and 30 000. Glycoprotein MFP II was not digested by bacterial collagenase. 7. The results suggest that elastic-fibre microfibrils are composed of a novel collagenous glycoprotein MFP I in association, as yet undefined, with a non-collagenous glycoprotein MFP II.