Regional correlation of emphysematous changes in lung function and structure: a comparison between pulmonary function testing and hyperpolarized MRI metrics

Regional and global relationships of lung function and structure were studied using hyperpolarized 3He MRI in a rat elastase-induced model of emphysema ( n = 4) and healthy controls ( n = 5). Fractional ventilation ( r) and apparent diffusion coefficient (ADC) of 3He were measured at a submillimeter planar resolution in ventral, middle, and dorsal slices 6 mo after model induction. Pulmonary function testing (PFT) was performed before MRI to yield forced expiratory volume in 50 ms (FEV50), airway resistance (RI), and dynamic compliance (Cdyn). Cutoff threshold values of ventilation and diffusion, r* and ADC*, were computed corresponding to 80% population of pixels falling above or below each threshold value, respectively. For correlation analysis, r* was compared with FEV50/functional residual capacity (FRC), RI and Cdyn, whereas ADC* was compared with FEV50/FRC, total lung capacity (TLC), and Cdyn. Regional correlation of r and ADC was evaluated by dividing each of the three lung slices into four quadrants. Cdyn was significantly larger in elastase rats (0.92 ± 0.16 vs. 0.61 ± 0.12 ml/cmH2O). The difference of RI and FEV50 was insignificant between the two groups. The r* of healthy rats was significantly larger than the elastase group (0.42 ± 0.03 vs. 0.28 ± 0.06), whereas ADC* was significantly smaller in healthy animals (0.27 ± 0.04 vs. 0.36 ± 0.01 cm2/s). No systematic difference in these quantities was observed between the three lung slices. A significant 33% increase in ADC* and a significant 31% decline in r* for elastase rats was observed compared with a significant 51% increase in Cdyn and a nonsignificant 26% decline in FEV50/FRC. Correlation of imaging and PFT metrics revealed that r and ADC divide the rats into two separate clusters in the sample space.

Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme

We recently described a chymostatin-sensitive elastase-2 as the major angiotensin (ANG) II-forming enzyme in the perfusate of the rat mesenteric arterial bed (MAB) with the same cDNA sequence as rat pancreatic elastase-2. The role of this enzyme in generating ANG II was examined in the rat isolated and perfused MAB. The vasoconstrictor effect elicited by ANG I and the renin substrate tetradecapeptide was only partially inhibited by captopril but abolished by the combination of captopril and chymostatin or N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK; inhibitor originally developed for human elastase-2). The effect induced by [Pro11,d-Ala12]-ANG I, an ANG I-converting enzyme (ACE)-resistant biologically inactive precursor of ANG II, was blocked by chymostatin or Ac-AAPL-CK. It was also demonstrated that cultured rat mesenteric endothelial cells synthesize elastase-2 and that mRNA for this enzyme can be detected in different rat tissues such as the pancreas, MAB, lung, heart, kidney, liver, and spleen. In conclusion, the demonstration of a functional alternative pathway to ACE for ANG II generation in the rat MAB and the fact that cultured MAB endothelial cells are capable of producing and secreting elastase-2 represent strong evidence of a physiological role for this enzyme in the rat vasculature.

The cell-specific elastase I enhancer comprises two domains

Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.

The cell-specific elastase I enhancer comprises two domains.

Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.

Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers

An elastase-human growth hormone (hGH) fusion gene containing 205 base pairs of elastase 5' flanking region is expressed exclusively in pancreatic acinar cells of transgenic mice. This paper shows that the promoter region (-72 to +8) and the enhancer (-205 to -73) function independently of each other. The elastase enhancer can activate the heterologous mouse metallothionein gene and the hGH gene promoters; conversely, enhancers from the thymocyte-specific murine leukemia virus MCF13 and the metal regulatory elements from the metallothionein gene can activate the elastase promoter in a variety of cell types. Combinations of immunoglobulin and elastase enhancers with a heterologous promoter and the hGH gene result in expression in all of the tissues predicted by the sum of each enhancer acting alone. Thus these enhancer elements act independently of each other, suggesting that they do not have silencing activity in cells in which they are normally inactive.

Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers.

An elastase-human growth hormone (hGH) fusion gene containing 205 base pairs of elastase 5' flanking region is expressed exclusively in pancreatic acinar cells of transgenic mice. This paper shows that the promoter region (-72 to +8) and the enhancer (-205 to -73) function independently of each other. The elastase enhancer can activate the heterologous mouse metallothionein gene and the hGH gene promoters; conversely, enhancers from the thymocyte-specific murine leukemia virus MCF13 and the metal regulatory elements from the metallothionein gene can activate the elastase promoter in a variety of cell types. Combinations of immunoglobulin and elastase enhancers with a heterologous promoter and the hGH gene result in expression in all of the tissues predicted by the sum of each enhancer acting alone. Thus these enhancer elements act independently of each other, suggesting that they do not have silencing activity in cells in which they are normally inactive.