Lung edema formation following inhalation injury: role of the bronchial blood flow

We investigated the contribution of the bronchial blood flow to the lung lymph flow (QL) and lung edema formation after inhalation injury in sheep (n = 18). The animals were equally divided into three groups and chronically prepared by implantation of cardiopulmonary catheters and a flow probe on the common bronchial artery. Groups 1 and 2 sheep were insufflated with 48 breaths of cotton smoke while group 3 received only room air. Just before injury, the bronchial artery of group 2 animals was occluded. The occlusion was maintained for the duration of the 24-h study period. At the end of the investigation, samples of lung were taken for determination of blood-free wet weight-to-dry weight ratio (W/D). Inhalation injury induced a sevenfold increase in QL in group 1 (7 +/- 1 to 50 +/- 9 ml/h; P less than 0.05) but only a threefold increase in group 2 (10 +/- 2 to 28 +/- 7 ml/h; P less than 0.05). The mean W/D value of group 1 animals was 23% higher than that of group 2 (5.1 +/- 0.4 vs. 3.9 +/- 0.2; P less than 0.05). Our data suggest that the bronchial circulation contributes to edema formation in the lung that is often seen after the acute lung injury with smoke inhalation.     

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans‐synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease‐associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP‐43, α‐synuclein, and the microtubule‐associated protein tau, can be driven out of the cell by an Hsc70 co‐chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.     

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.     

Systemic blood flow to sheep lung: comparison of flow probes and microspheres.

Discrepancies exist between experimental measurements of the systemic blood flow to sheep lung by use of microsphere techniques and flow probes on the bronchial artery. In these studies, we simultaneously measured the blood flow through the bronchial artery, using a transit time flow probe, and the systemic blood flow to left lung, using radioactive microspheres. All measurements were made on conscious sheep previously prepared with chronic catheterizations of the left atrium, aorta, and vena cava and a flow probe around the bronchial artery. Inflatable occluder cuffs were placed around the pulmonary and bronchoesophageal arteries. Bronchial artery blood flow in six sheep was 25.3 +/- 5.2 ml/min or 0.4% of the cardiac output. Systemic blood flow to left lung, measured with microspheres, was 54.1 +/- 14.2 ml/min. Calculated systemic blood flow to that portion of sheep lung perfused by the bronchial artery was 127.6 +/- 35.3 ml/min or 1.9% of cardiac output. Occlusion of the bronchoesophageal artery reduced bronchial artery flow to near zero, whereas total systemic blood to the lung was reduced by only 55%. Blood flow to the intraparenchymal cartilaginous airways was reduced 60-90% after occlusion of the bronchoesophageal artery. Sheep, like most mammals, have multiple and complex systemic arterial inputs to the lungs. We conclude that multiple branches of the bronchoesophageal artery provide most but not all of the systemic blood flow to the intraparenchymal cartilaginous airways but that over one-half of the total systemic blood flow to sheep lung comes from sources other than the common bronchial artery.     

Pulmonary edema with smoke inhalation, undetected by indicator-dilution technique

Despite experimental evidence for an increase in extravascular lung water (EVLW) after inhalation injury, thermal-dye estimations of EVLW, extravascular thermal volume (EVTV), have repeatedly failed to demonstrate its presence in patients. This situation was evaluated in a sheep model. Under halothane anesthesia one lung was insufflated with cotton smoke and the other with air. EVTV values were 8.4 +/- 0.48 ml/kg at base line and were not elevated at 24 h after smoke inhalation (8.3 +/- 0.45 ml/kg; means +/- SE). Gravimetric analysis 24 h after smoke inhalation showed the development of edema in smoke-exposed lungs. The blood-free wet weight-to-dry weight ratio of the smoke-exposed lungs (5.4 +/- 0.32) was significantly higher compared with the contralateral unsmoked lungs (4.3 +/- 0.15; P less than or equal to 0.05). The thermal-dye technique thus underestimates EVLW. Poor perfusion of the smoke-exposed lungs 24 h after injury was demonstrated indirectly by killing a group of sheep with T-61, an agent that causes a dark red coloration of well-perfused lung areas, as well as directly by measurement of blood flow utilizing a radiolabeled microsphere technique. Thus the inability of the thermal-dye technique to detect the lung edema may be the result of poor perfusion of the injured lung.