The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actinassociated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo R cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo R cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinaseinduced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.Leukocytes are mobile cells and their actin cytoskeleton plays a central role in the locomotion, transmigration, and phagocytosis. These activities are essential for the function of myeloid cells, including neutrophils, monocytes, and macrophages, in defensive and autoimmune responses (1). Despite the significant biological and medical importance, the regulation of actin cytoskeleton in myeloid cells is not well understood.Calponin is an actin filament-associated protein of 34 -37 kDa (292-330 amino acids) found in smooth muscle (2) as well as non-muscle cells (3,4). Through high affinity binding to F-actin, calponin inhibits the actin-activated myosin MgATPase (5-8) and motor activity (9 -11). The association of calponin with actin filaments and its regulatory function have led to a model wherein calponin may represent a thin filament regulatory mechanism modulating smooth muscle contraction (12).Three isoforms of calponin (h1, h2, and acidic) have been identified in higher vertebrates as the products of three homologous genes (13-17). The three isoforms of calponin have distinct theoretical isoelectric points (pI values): H1-calponin is basic (pI 9.4), h2-calponin is near neutral (pI 7.5), whereas the acidic calponin has a lower pI of 5.2. H1-calponin is the predominant isoform specifically expressed in differentiated smooth muscle cells and its role in regulating smooth muscle contractility (9) is a focus of ongoing research. The majority of previous structural and functional studies of calponin were obtained from chicken gizzard calponin that is equivalent to the mammalian h1-calponin. The acidic c...
Mokres LM, Parai K, Hilgendorff A, Ertsey R, Alvira CM, Rabinovitch M, Bland RD. Prolonged mechanical ventilation with air induces apoptosis and causes failure of alveolar septation and angiogenesis in lungs of newborn mice.
Rationale: Mechanical ventilation with O 2 -rich gas (MV-O 2 ) offers life-saving treatment for respiratory failure, but also promotes lung injury. We previously reported that MV-O 2 of newborn mice increased lung elastase activity, causing elastin degradation and redistribution of elastic fibers from septal tips to alveolar walls. These changes were associated with transforming growth factor (TGF)-b activation and increased apoptosis leading to defective alveolarization and lung growth arrest, as seen in neonatal chronic lung disease. Objectives: To determine if intratracheal treatment of newborn mice with the serine elastase inhibitor elafin would prevent MV-O 2 -induced lung elastin degradation and the ensuing cascade of events causing lung growth arrest. Methods: Five-day-old mice were treated via tracheotomy with recombinant human elafin or vehicle (lactated-Ringer solution), followed by MV with 40% O 2 for 8-24 hours; control animals breathed 40% O 2 without MV. At study's end, lungs were harvested to assess key variables noted below. Measurements and Main Results: MV-O 2 of vehicle-treated pups increased lung elastase and matrix metalloproteinase-9 activity when compared with unventilated control animals, causing elastin degradation (urine desmosine doubled), TGF-b activation (pSmad-2 tripled), and apoptosis (cleaved-caspase-3 increased 10-fold). Quantitative lung histology showed larger and fewer alveoli, greater inflammation, and scattered elastic fibers. Elafin blocked these MV-O 2 -induced changes. Conclusions: Intratracheal elafin, by blocking lung protease activity, prevented MV-O 2 -induced elastin degradation, TGF-b activation, apoptosis, and dispersion of matrix elastin, and attenuated lung structural abnormalities noted in vehicle-treated mice after 24 hours of MV-O 2 . These findings suggest that elastin breakdown contributes to defective lung growth in response to MV-O 2 and might be targeted therapeutically to prevent MV-O 2 -induced lung injury.
Mechanical ventilation (MV) with O(2)-rich gas (MV-O(2)) offers life-saving treatment for newborn infants with respiratory failure, but it also can promote lung injury, which in neonates translates to defective alveolar formation and disordered lung elastin, a key determinant of lung growth and repair. Prior studies in preterm sheep and neonatal mice showed that MV-O(2) stimulated lung elastase activity, causing degradation and remodeling of matrix elastin. These changes yielded an inflammatory response, with TGF-β activation, scattered elastic fibers, and increased apoptosis, culminating in defective alveolar septation and arrested lung growth. To see whether sustained inhibition of elastase activity would prevent these adverse pulmonary effects of MV-O(2), we did studies comparing wild-type (WT) and mutant neonatal mice genetically modified to express in their vascular endothelium the human serine elastase inhibitor elafin (Eexp). Five-day-old WT and Eexp mice received MV with 40% O(2) (MV-O(2)) for 24-36 h. WT and Eexp controls breathed 40% O(2) without MV. MV-O(2) increased lung elastase and MMP-9 activity, resulting in elastin degradation (urine desmosine doubled), TGF-β activation (pSmad-2 increased 6-fold), apoptosis (cleaved-caspase-3 increased 10-fold), and inflammation (NF-κB activation, influx of neutrophils and monocytes) in lungs of WT vs. unventilated controls. These changes were blocked or blunted during MV-O(2) of Eexp mice. Scattered lung elastin and emphysematous alveoli observed in WT mice after 36 h of MV-O(2) were attenuated in Eexp mice. Both WT and Eexp mice showed defective VEGF signaling (decreased lung VEGF-R2 protein) and loss of pulmonary microvessels after lengthy MV-O(2), suggesting that elafin's beneficial effects during MV-O(2) derived primarily from preserving matrix elastin and suppressing lung inflammation, thereby enabling alveolar formation during MV-O(2). These results suggest that degradation and remodeling of lung elastin can contribute to defective lung growth in response to MV-O(2) and might be targeted therapeutically to prevent ventilator-induced neonatal lung injury.
Elastin plays a pivotal role in lung development. We therefore queried if elastin haploinsufficient newborn mice ( Eln+/−) would exhibit abnormal lung structure and function related to modified extracellular matrix (ECM) composition. Because mechanical ventilation (MV) has been linked to dysregulated elastic fiber formation in the newborn lung, we also asked if elastin haploinsufficiency would accentuate lung growth arrest seen after prolonged MV of neonatal mice. We studied 5-day-old wild-type ( Eln+/+) and Eln+/− littermates at baseline and after MV with air for 8–24 h. Lungs of unventilated Eln+/− mice contained ∼50% less elastin and ∼100% more collagen-1 and lysyl oxidase compared with Eln+/+ pups. Eln+/− lungs contained fewer capillaries than Eln+/+ lungs, without discernible differences in alveolar structure. In response to MV, lung tropoelastin and elastase activity increased in Eln+/+ neonates, whereas tropoelastin decreased and elastase activity was unchanged in Eln+/− mice. Fibrillin-1 protein increased in lungs of both groups during MV, more in Eln+/− than in Eln+/+ pups. In both groups, MV caused capillary loss, with larger and fewer alveoli compared with unventilated controls. Respiratory system elastance, which was less in unventilated Eln+/− compared with Eln+/+ mice, was similar in both groups after MV. These results suggest that elastin haploinsufficiency adversely impacts pulmonary angiogenesis and that MV dysregulates elastic fiber integrity, with further loss of lung capillaries, lung growth arrest, and impaired respiratory function in both Eln+/+ and Eln+/− mice. Paucity of lung capillaries in Eln+/− newborns might help explain subsequent development of pulmonary hypertension previously reported in adult Eln+/− mice.
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