Daniela Negrini

The sensitivity of versican from rabbit lung to gelatinase A (MMP‐2) and B (MMP‐9) and its involvement in the development of hydraulic lung edema

Large chondroitinsulphate‐containing proteoglycan (versican) isolated from rabbit lung was cleaved by purified gelatinase A (MMP‐2) and gelatinase B (MMP‐9), as well as by crude enzyme extract from rabbit lung with hydraulic edema. Gelatine zymography, performed after purification of gelatinases by affinity chromatography, demonstrated that the enzyme extract contained two main gelatinolytic bands at about 92 kDa and 72 kDa, identified by specific antisera as the latent proMMP‐9 and proMMP‐2, respectively. Moreover, enzyme extract from edematous lung showed an increased amount of the proteolytically activated forms of both gelatinases with respect to normal controls. These results suggest that MMP‐2 and MMP‐9 are involved in the breakdown of versican occurring in rabbit lung during the development of hydraulic edema.

Subatmospheric pressure in the rabbit pleural lymphatic network

1 Hydraulic pressure in intercostal and diaphragmatic lymphatic vessels was measured through the micropuncture technique in 23 anaesthetised paralysed rabbits. Pleural lymphatic vessels with diameters ranging from 55 to 950 μm were observed under stereomicroscope view about 3–4 h after intrapleural injection of 20 % fluorescent dextrans. 2 Lymphatic pressure oscillated from a minimum (Pmin) to a maximum (Pmax) value, reflecting oscillations in phase with cardiac activity (cardiogenic oscillations) and lymphatic myogenic activity. With intact pleural space, Pmin in submesothelial diaphragmatic lymphatic vessels of the lateral apposition zone was −9.1 ± 4.2 mmHg, more subatmospheric than the simultaneously recorded pleural liquid pressure amounting to −3.9 ± 1.2 mmHg. In extrapleural intercostal lymphatic vessels Pmin averaged −1.3 ± 2.7 mmHg. 3 Cardiogenic pressure oscillations (Pmax−Pmin), were observed in all recordings; their mean amplitude was about 5 mmHg and was not dependent upon frequency of cardiac contraction, nor lymphatic vessel diameter, nor the Pmin value. 4 Intrinsic contractions of lymphatic vessel walls caused spontaneous pressure waves of about 7 mmHg in amplitude at a rate of 8 cycles min−1. 5 These results demonstrated the ability of pleural lymphatic vessels to generate pressure oscillations driving fluid from the subatmospheric pleural space into the lymphatic network.

Involvement of lung interstitial proteoglycans in development of hydraulic- and elastase-induced edema

We extracted and isolated proteoglycans from lung tissue samples obtained from three groups of anesthetized rabbits: 1) control animals (C; n= 8) killed by overdose after 180 min; 2) animals receiving an intravenous saline infusion (S; n = 4, 1.5 ml ⋅ kg-1 ⋅ min-1) for 180 min; 3) animals receiving an intravenous bolus of 200 μg of pancreatic elastase (E; n = 4), killed after 200 min. The lung dry weight-to-wet weight ratio in the three groups was 5.2 ± 0.2, 6.0 ± 0.4, and 5.6 ± 0.5, respectively. Gel-filtration analysis showed a massive fragmentation of the versican family of the extracellular matrix (ECM) in the S groups and a marked degradation of heparan sulfate-containing proteoglycans, including perlecan of the basement membrane, in the E group. The binding properties of total proteoglycans to other ECM components were lowered in both groups relative to control. The decrease in proteoglycan binding was more pronounced for collagen type IV in the E group relative to C (-93.5%, P < 0.05) and for hyaluronic acid in the S groups (-85.8%, P < 0.05). These findings suggest that elastase treatment produces a major degree of damage to the organization of basement membrane, whereas saline loading affects more markedly the architecture of interstitial ECM. Qualitative zymography performed on lung extracts showed increased gelatinase activities in both S and E groups, providing direct evidence that the activation of tissue proteinases may play a role in acute lung injury.

Proteoglycan involvement during development of lesional pulmonary edema

We evaluated the effect of pancreatic elastase (7 IU i.v.) on pulmonary interstitial pressure (Pip) in in situ rabbit lungs by a micropuncture technique through the intact parietal pleura. Pip was -10.8 +/- 2.2 (SD) cmH2O in the control condition, increased to +5.1 +/- 1.7 cmH2O at approximately 60 min [condition referred to as mild edema (ME)], and subsequently decreased to -0.15 +/- 0.8 cmH2O, remaining steady from 80 up to 200 min with a marked increase in lung wet-to-dry weight ratio [condition referred to as severe edema (SE)], suggesting an increase in tissue compliance. We functionally correlated the measured Pip to structural modifications of proteoglycans, the major interfibrillar component of the extracellular matrix (ECM). The strength of the noncovalent bonds linking proteoglycans to other ECM components decreased with increasing severity of edema, as indicated by the increased extractability of proteoglycans with guanidine hydrochloride. Total proteoglycan recovery (expressed as microgram hexuronate/g dry tissue) increased from 436.8 +/- 14 in the control condition to 495.3 +/- 23 and 547.0 +/- 10 in ME and SE, respectively. Gel-filtration chromatography showed in ME a fragmentation of heparan sulfate proteoglycans, suggesting that elastase treatment first affected basement membrane integrity, whereas large chondroitin sulfate proteoglycans were degraded only in SE. Elastase caused a fragmentation only of the core protein of proteoglycans, the binding properties of which to collagens, fibronectin, and hyaluronic acid were markedly decreased, as indicated by a solid-phase binding assay. The sequential degradation of heparan sulfate and chondroitin sulfate proteoglycans may account for the initial increase in microvascular permeability, followed by a loss of the native architecture of the ECM, which may be responsible for the increase in tissue compliance.

Lymphatic anatomy and biomechanics

Abstract  Lymph formation is driven by hydraulic pressure gradients developing between the interstitial tissue and the lumen of initial lymphatics. While in vessels equipped with lymphatic smooth muscle cells these gradients are determined by well‐synchronized spontaneous contractions of vessel segments, initial lymphatics devoid of smooth muscles rely on tissue motion to form lymph and propel it along the network. Lymphatics supplying highly moving tissues, such as skeletal muscle, diaphragm or thoracic tissues, undergo cyclic compression and expansion of their lumen imposed by local stresses arising in the tissue as a consequence of cardiac and respiratory activities. Active muscle contraction and not passive tissue displacement is required to support an efficient lymphatic drainage, as suggested by the fact that the respiratory activity promotes lymph formation during spontaneous, but not mechanical ventilation. The mechanical properties of the lymphatic wall and of the surrounding tissue also play an important role in lymphatic function. Modelling of stress distribution in the lymphatic wall suggests that compliant vessels behave as reservoirs accommodating absorbed interstitial fluid, while lymphatics with stiffer walls, taking advantage of a more efficient transmission of tissue stresses to the lymphatic lumen, propel fluid through the lumen of the lymphatic circuit.

Pleural liquid pressure over the interlobar mediastinal and diaphragmatic surfaces of the lung

Pleural liquid pressure (Pliq) was measured in the interlobar fissures and over the mediastinal apical, cardiac and diaphragmatic surface. When compared to costal values, Pliq was more subatmospheric over the cardiac region at all lung heights by about 5 cm H2O so that a costo-hilar gradient down the interlobar fissures of about 0.7 cm H2O/cm was found. No differences were found for other surfaces. Cardiac values, unlike costal ones, became more subatmospheric on increasing ventilation and heart-rate. On the basis of a Starling equilibrium a costo-hilar gradient of Pliq as well as more negative values in the cardiac surface can be explained if hydrostatic pressure in visceral pleura and/or hydraulic conductivity of parietal pleura are lower on mediastinal as compared to costal side. However the ventilation and heart-beat dependence of Pliq on cardiac region suggest a possible role of lymphatic in setting pleural liquid pressure.

Extracellular matrix and mechanical ventilation in healthy lungs: back to baro/volotrauma?

Purpose of reviewThe extracellular matrix plays an important role in the biomechanical behaviour of the lung parenchyma. The matrix is composed of a three-dimensional fibre mesh filled with different macromolecules, including proteoglycans which have important functions in many lung pathophysiological processes, as they regulate tissue hydration, macromolecular structure and function, response to inflammatory agents, and tissue repair and remodelling. The aim of this review is to describe the role of mechanical ventilation on pulmonary extracellular matrix structure and function. Recent findingsRecent experimental and clinical data suggest that in healthy lungs, mechanical ventilation with tidal volume ranging between 7 and 12 ml/kg in the absence of positive end-expiratory pressure may lead to endothelial, extracellular matrix and peripheral airways damage without major inflammatory response. Several mechanisms may explain damage to the lung structure induced by mechanical ventilation: regional overdistension, ‘low lung volume’ associated with tidal airway closure, and inactivation of surfactant. SummaryTidal volume reduction to 6 ml/kg may be useful during mechanical ventilation of healthy lungs. The study of the extracellular matrix may be useful to better understand the pathophysiology of ventilator-induced lung injury in healthy and diseased lungs.

The extracellular matrix of the lung and its role in edema formation

The extracellular matrix is composed of a three-dimensional fiber mesh filled with different macromolecules such as: collagen (mainly type I and III), elastin, glycosaminoglycans, and proteoglycans. In the lung, the extracellular matrix has several functions which provide: 1) mechanical tensile and compressive strength and elasticity, 2) low mechanical tissue compliance contributing to the maintenance of normal interstitial fluid dynamics, 3) low resistive pathway for an effective gas exchange, d) control of cell behavior by the binding of growth factors, chemokines, cytokines and the interaction with cell-surface receptors, and e) tissue repair and remodeling. Fragmentation and disorganization of extracellular matrix components comprises the protective role of the extracellular matrix, leading to interstitial and eventually severe lung edema. Thus, once conditions of increased microvascular filtration are established, matrix remodeling proceeds fairly rapidly due to the activation of proteases. Conversely, a massive matrix deposition of collagen fiber decreases interstitial compliance and therefore makes the tissue safety factor stronger. As a result, changes in lung extracellular matrix significantly affect edema formation and distribution in the lung.

Role of the diaphragm in setting liquid pressure in serous cavities

Supraphrenic and subphrenic liquid pressures in supine dogs and rabbits were found to be about 2 cm H2O more negative than costal and mediastinal liquid pressures at the same lung height. Peritoneal liquid pressure was negative at all lung heights in dogs and slightly positive below 25% of lung height in rabbits. The vertical gradients of liquid pressure did not differ significantly in either species between the pleural and peritoneal cavities and were lower than 1 cm H2O/cm. From the peritoneal protein concentration (2.3 and 3.5 g/100 ml in dogs and rabbits, respectively) we calculated a filtration pressure for the peritoneal mesothelium and a lymphatic role was hypothesized to explain negative liquid pressures in the peritoneal cavity. The greater negativity of the subphrenic as compared with the peritoneal data could be related to a strong respiratory-dependent lymphatic action. A similar mechanism and/or a low filtration conductance of the diaphragmatic pleura may explain the costo-supraphrenic liquid pressure difference. The present results, extending previously published data on the mediastinal side of the lung, further support the existence of local differences in fluid dynamics in the pleural cavity.

Pulmonary Microvascular Pressure Profile During Development of Hydrostatic Edema

Objective: To measure, in intact closed chest, the pressure in the pulmonary microvasculature during transition to mild interstitial edema.