Joseph M. Cerreta

Decreased elastic fibers and desmosine content in incompetent cervix

Incompetence of the uterine cervix is a syndrome of painless, progressive dilatation and effacement occurring between the sixteenth and twenty-fourth weeks of gestation that represents abnormal functioning. It may serve as a model to elucidate normal function. Because the incompetent cervix results in painless opening of this organ without uterine contraction before term gestation, it is considered one of the causes of midtrimester spontaneous abortion, habitual spontaneous abortion, and early preterm labor. Untreated, it leads to rapid expulsion and often death of the fetus. We used light microscopy to compare decreased elastic fibers in incompetent cervices with those of normal nonpregnant and pregnant cervices. Morphologic analysis of this difference was extended to biochemical quantification of elastin content in one patient with cervical incompetence. The decrease in elastin suggests that one function of cervical elastin may be to maintain a closed and undilated cervix throughout gestation. There may be a relationship between changes in cross-linked elastin and the incompetent cervix; further studies are therefore indicated.

AEROSOLIZED HYALURONAN LIMITS AIRSPACE ENLARGEMENT IN A MOUSE MODEL OF CIGARETTE SMOKE–INDUCED PULMONARY EMPHYSEMA

This study was designed to determine if aerosolized hyaluronan (HA) could prevent airspace enlargement and elastic fiber injury in a mouse model of cigarette smoke–induced pulmonary emphysema. Compared to untreated/smoked controls, HA-treated animals showed statistically significant reductions in mean linear intercept (54 versus 65 μm; P < .001) and elastic fiber breakdown products (desmosine and isodesmosine) in bronchoalveolar lavage fluid (0.3 versus 7.0 ng/mL; P < .05). As in previous studies, the aerosolized HA showed preferential binding to elastic fibers, suggesting that it may protect them from injury. These findings support further investigation of the potential use of HA as a treatment for pulmonary emphysema.

Modulation of airspace enlargement in elastase-induced emphysema by intratracheal instillment of hyaluronidase and hyaluronic acid

The study examined how lung hyaluronic acid content influences airspace enlargement in elastase-induced emphysema. To determine the effect of a decrease in hyaluronic acid, hamsters received a single intratracheal instillment of hyaluronidase 24 h prior to administration of pancreatic elastase by the same route. One week later, these animals showed significantly greater airspace enlargement than controls sequentially instilled with saline and elastase (128 vs. 100 microns; p < .05). Conversely, intratracheal administration of hyaluronic acid immediately after elastase instillment resulted in a marked decrease in airspace enlargement at 1 week compared to controls receiving elastase followed by saline (82 vs. 122 microns; p = .005). Since hyaluronic acid has no elastase inhibitory capacity, its effect may involve extracellular matrix interactions not directly related to elastic fiber breakdown. This concept is supported by the finding that animals treated with hyaluronidase and elastase showed no greater loss of lung elastin than that observed in the saline/elastase control group, despite demonstrating a marked increase in airspace enlargement. Further work is needed to determine how hyaluronic acid influences airspace enlargement and to evaluate the potential use of this substance as a treatment for emphysema.

Amiodarone-Induced Pulmonary Fibrosis in Hamsters

Amiodarone, a cardiac antiarrhythmic agent, has been associated with the development of interstitial pulmonary fibrosis in patients receiving prolonged therapy with the drug. To further assess the toxic effects of amiodarone on lung tissue, Syrian hamsters were given a single intratracheal insufflation of the agent and evaluated for histologic evidence of lung injury. Control animals received intratracheal insufflations of the vehicle in which amiodarone was dissolved. After an initial, transient alveolitis in both experimental and control animals, the amiodarone-treated lungs developed increased interstitial thickening due to fibrinous exudates, alveolar epithelial hyperplasia, inflammatory cell infiltrates, and marked deposition of collagen manifested on trichrome staining. Controls, in contrast, showed nearly complete resolution of the initial alveolitis. An unusual feature of the amiodarone-induced lung injury was reemergence of the alveolitis between 5 and 14 days, which included a marked influx of eosinophils into the lung. Although the precise mechanism of the lung injury is not known, the persistence of the acute inflammatory cells as well as the presence of eosinophils suggests a hypersensitivity-type reaction. Furthermore, the progression of lung injury to fibrosis after a single insult with the drug suggests that mere discontinuation of amiodarone therapy in humans may not reverse the disease process, but that corticosteroid therapy may also be required. Amiodarone appears to be a useful agent to induce diffuse fibrotic reactions in the lung that morphologically resemble idiopathic pulmonary fibrosis in humans.

Further Investigation of the Use of Intratracheally Administered Hyaluronic Acid to Ameliorate Elastase-Induced Emphysema

Previously, this laboratory has shown that intratracheally administered hyaluronic acid (HA) significantly reduces air-space enlargement in a hamster model of emphysema induced with pancreatic elastase. Whereas HA was given immediately following elastase in those initial studies, the current investigation determined the effect of instilling HA up to 2 h before or after intratracheal administration of elastase to hamsters. Both 1 and 2 mg HA, given 2 h before pancreatic elastase, significantly decreased (p < .05) air-space enlargement compared to controls (as measured by the mean linear intercept). Instillment of 2 mg HA, 1 h after pancreatic elastase, had a similar effect (p < .05). In contrast, 1 mg HA, given 1 or 2 h after pancreatic elastase, did not significantly affect the mean linear intercept. Against human neutrophil elastase, HA exhibited the same protective effect. While neutrophil elastase induced less air-space enlargement than pancreatic elastase, both 1 and 4 mg of HA, given 2 h prior to the enzyme, still produced a significant reduction (p < .05) in the mean linear intercept. HA exerted this effect despite the fact that it initiates a transient influx of neutrophils into the lung. Since HA does not slow the clearance of intratracheally instilled [14C] albumin from the lung, its mechanism of action may not involve physical interference with the movement of elastase through the lung, but may instead depend on interaction with elastic fibers. Evidence for an association between these two matrix constituents was provided by studies using fluorescein-labeled HA. Overall, these results further suggest that HA may be useful in preventing lung injury by elastases.

Synthesis of crosslinked elastin by an endothelial cell culture

Summary Synthesis of crosslinked elastin by a major lung cell has not previously been reported. Elastin production by an established clone of rat lung endothelial cells was detected by two separate, highly sensitive methods. The first procedure involved isolation and identification of the labelled, elastin-specific crosslinking amino acids desmosine and isodesmosine by thin layer electrophoresis and radioautography. The second procedure involved detection of elastin by immunofluorescence, using anti-rat lung elastin peptide serum.

Orientation of elastic fibers in the human cervix

We have determined the microanatomy of the cervix in relation to elastic fibers by serial dissection of human cervix in three tissue planes: cross, sagittal, and frontal sections. Analysis suggests that elastin is localized to specific regions of the uterine cervix and not dispersed throughout the connective tissue stroma. By Musto stain the majority of elastic fibers are noted to be oriented from the external os to the periphery and from there in a band upward toward the internal os where they become sparse in the area of the cervix with the greatest amount of smooth muscle just below the internal os. Elastic fibers were noted to be sparsely distributed in the cervical stroma.

The content of elastin in the uterine cervix

Mature, crosslinked elastin has been isolated from 4 human and 12 monkey uterine cervices. A modification of previous methods for determination of elastin content was devised to quantitate the low amounts of elastin in the crude connective tissue of uterine cervices. The percentage of elastin was found to range between 0.9 and 2.4% and did not appear to change at various stages of gestation.

Pulmonary Air-Space Enlargement Induced by Intratracheal Instillment of Hyaluronidase and Concomitant Exposure to 60% Oxygen

Although emphysema is generally characterized by damage to pulmonary elastic fibers, the causes of such injury appear to be complex and are not entirely explained by a singular imbalance between elastases and their inhibitors. Other factors could compromise elastic fiber integrity. To test the validity of this argument, hamsters were instilled intratracheally with a nonelastolytic enzyme, hyaluronidase (which reduces lung hexuronic acid content by 21% after 24 h), then exposed to an otherwise nontoxic concentration of oxygen (60%) for 4 days. Additional groups were given (1) hyaluronidase and room air, (2) saline and 60% oxygen, and (3) saline and room air. Treatment with both hyaluronidase and 60% oxygen resulted in a significant increase in air-space enlargement at 4 days (67.1 vs. 57.9 microns for saline/room air controls; p < .05), which was accompanied by only minimal inflammatory changes, as determined by both light microscopy and lavage cytology. Animals receiving either hyaluronidase or 60% oxygen alone showed no significant increases in air-space size compared to those given saline and exposed to room air. While the mechanisms responsible for these results are unclear, the marked increase in radiolabeling of lung elastin cross-links (desmosine and isodesmosine) in animals receiving both hyaluronidase and 60% oxygen (429 vs. 168 cpm/g dry lung for saline/room air controls; p < .05), as well as a significant decrease in total lung desmosine and isodesmosine (32.5 vs. 37.7 micrograms/lung for saline/room air controls; p < .05), suggests that elastic fiber damage is a potential factor. Moreover, only those animals receiving both hyaluronidase and 60% oxygen showed a significant rise in cell-free elastase activity in lavage fluids compared to saline/room air controls (83.3 vs. 48.3 ng; p < .05). On the basis of these findings, it is concluded that while elastic fiber damage may be a common pathway in emphysema, the factors that initiate the disease may be more varied than previously suspected and not always related to the balance between elastases and their inhibitors.

Conclusive evidence for the presence of elastin in human and monkey cervix

Mature, cross-linked elastin has been isolated from human and monkey uterine cervix and characterized by the presence of the specific amino acid markers, desmosine and isodesmosine. These two amino acids could be detected in human uterine cervical tissue derived from pregnant and nonpregnant subjects as well as from monkey cervical tissue. These findings provide conclusive evidence that elastin, a protein which is the major constituent of elastic tissue, is present in the cervix. It is suggested on theoretical grounds that elastin has a role to play in the dilatation of the uterine cervix during parturition.