Alisha M. Gruntman

Cellular kinetics and modeling of bronchioalveolar stem cell response during lung regeneration.

Organ regeneration in mammals is hypothesized to require a functional pool of stem or progenitor cells, but the role of these cells in lung regeneration is unknown. Whereas postnatal regeneration of alveolar tissue has been attributed to type II alveolar epithelial cells (AECII), we reasoned that bronchioalveolar stem cells (BASCs) have the potential to contribute substantially to this process. To test this hypothesis, unilateral pneumonectomy (PNX) was performed on adult female C57/BL6 mice to stimulate compensatory lung regrowth. The density of BASCs and AECII, and morphometric and physiological measurements, were recorded on days 1, 3, 7, 14, 28, and 45 after surgery. Vital capacity was restored by day 7 after PNX. BASC numbers increased by day 3, peaked to 220% of controls (P<0.05) by day 14, and then returned to baseline after active lung regrowth was complete, whereas AECII cell densities increased to 124% of baseline (N/S). Proliferation studies revealed significant BrdU uptake in BASCs and AECII within the first 7 days after PNX. Quantitative analysis using a systems biology model was used to evaluate the potential contribution of BASCs and AECII. The model demonstrated that BASC proliferation and differentiation contributes between 0 and 25% of compensatory alveolar epithelial (type I and II cell) regrowth, demonstrating that regeneration requires a substantial contribution from AECII. The observed cell kinetic profiles can be reconciled using a dual-compartment (BASC and AECII) proliferation model assuming a linear hierarchy of BASCs, AECII, and AECI cells to achieve lung regrowth.

Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AATs antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

Matrix modulation of compensatory lung regrowth and progenitor cell proliferation in mice

Mechanical stress is an important modulator of lung morphogenesis, postnatal lung development, and compensatory lung regrowth. The effect of mechanical stress on stem or progenitor cells is unclear. We examined whether proliferative responses of epithelial progenitor cells, including dually immunoreactive (CCSP and proSP-C) progenitor cells (CCSP+/SP-C+) and type II alveolar epithelial cells (ATII), are affected by physical factors found in the lung of emphysematics, including loss of elastic recoil, reduced elastin content, and alveolar destruction. Mice underwent single lung pneumonectomy (PNY) to modulate transpulmonary pressure (mechanical stress) and to stimulate lung regeneration. Control mice underwent sham thoracotomy. Plombage of different levels was employed to partially or completely abolish this mechanical stress. Responses to graded changes in transpulmonary pressure were assessed in elastin-insufficient mice (elastin +/-, ELN+/-) and elastase-treated mice with elastase-induced emphysema. Physiological regrowth, morphometry (linear mean intercept; Lmi), and the proliferative responses of CCSP+/SP-C+, Clara cells, and ATII were evaluated. Plombage following PNY significantly reduced transpulmonary pressure, regrowth, and CCSP+/SP-C+, Clara cell, and ATII proliferation following PNY. In the ELN+/- group, CCSP+/SP-C+ and ATII proliferation responses were completely abolished, although compensatory lung regrowth was not significantly altered. In contrast, in elastase-injured mice, compensatory lung regrowth was significantly reduced, and ATII but not CCSP+/SP-C+ proliferation responses were impaired. Elastase injury also reduced the baseline abundance of CCSP+/SP-C+, and CCSP+/SP-C+ were found to be displaced from the bronchioalveolar duct junction. These data suggest that qualities of the extracellular matrix including elastin content, mechanical stress, and alveolar integrity strongly influence the regenerative capacity of the lung, and the patterns of cell proliferation in the lungs of adult mice.

Age-Dependent Decline in Mouse Lung Regeneration with Loss of Lung Fibroblast Clonogenicity and Increased Myofibroblastic Differentiation

While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration.

Age Dependence of Lung Mesenchymal Stromal Cell Dynamics Following Pneumonectomy

Aging is a critical determinant of regenerative capacity in many organ systems, but it remains unresolved in the lung. This study examines murine lung cell dynamics during age-dependent lung regeneration. Proliferation of lung progenitor cells (EpCAM(neg)/Sca-1(high) lung mesenchymal stromal cells - LMSCs, EpCAM(pos)/Sca-1(low) epithelial progenitor cells, proSP-C(pos) alveolar type II epithelial cells - AECII, and CD31(pos) - endothelial cells) was tracked to day 3 or 7 after pneumonectomy (PNX) or SHAM surgery in 3, 9, and 17 month mice. In 3 month mice, post-PNX LMSC proliferation peaked early (3 days), with 50%-80% more BrdU-positive cells than the other cell types, which peaked later (4-7 days). In older mice (9 and 17 month), abundance and post-PNX proliferation of LMSCs at day 3 were reduced (40%-80%). In both young and old mice, LMSCs were isolated and compared phenotypically with whole lung non-LMSCs. Donor age had no qualitative effect on the phenotype (LMSC vs. non-LMSC), with increased expression of CD90/Thy1, CD105/Eng, CD106/Vcam, CD146/Mcam, and Pdgfrα, and up-regulation of mRNA encoding Fap, Eln, Col1a1, Col3a1, Aldh1a3, Arhgef25, Dner, Fgfr1, and Midkine. However, compared with LMSCs isolated from young mice, LMSCs from older mice exhibited reduced mRNA expression of retinoic acid (Aldh1a3, Rbp4), Fgf/Wnt (Fgfr1, Sfrp1, Wnt2, and Ctnnb1), and elastogenesis (Col1a1, Eln, Fbn1, and Sdc2) pathway genes. Isolated LMSCs from older mice also demonstrated lower colony-forming units (-67%), growth potential (-60% by day 7), ALDH activity (-49%), and telomerase activity (-47%). Therefore, age is associated with declining proliferative potential and regenerative functions of LMSCs in the lung.

A Multicenter Retrospective Study of 151 Renal Biopsies in Horses

BACKGROUND Renal biopsies are uncommonly performed in horses and little is known about their diagnostic utility and associated complication rate. OBJECTIVE To describe the techniques, the complication rate, risk factors, and histopathology results; as well as evaluate the safety and diagnostic utility of renal biopsy in the horse. ANIMALS One hundred and forty-six horses from which 151 renal biopsies were obtained. Animals ranged in age from 48 hours to 30 years. METHODS Multicenter retrospective study, with participation of 14 institutions (1983-2009). RESULTS Renal biopsy in horses was associated with a similar rate of complications (11.3%) to that occurring in humans and companion animals. Complications were generally associated with hemorrhage or signs of colic, and required treatment in 3% of cases. Fatality rate was low (1/151; 0.7%). Biopsy specimens yielded sufficient tissue for a histopathologic diagnosis in most cases (94%) but diagnoses had only fair (72%) agreement with postmortem findings. Risk factors for complications included biopsy specimens of the left kidney (P = .030), a diagnosis of neoplasia (P = .004), and low urine specific gravity (P = .030). No association with complications was found for age, sex, breed, institution, presenting complaint, other initial clinicopathologic data, biopsy instrument, needle size, or use of ultrasonographic guidance. CONCLUSIONS AND CLINICAL IMPORTANCE Renal biopsy in horses has low morbidity and results in a morphological histopathologic diagnosis in 94% of cases. However, this procedure might result in serious complications and should only be used when information obtained would be likely to impact decisions regarding patient management and prognosis.

A Comparison of Total, Respirable, and Real-Time Airborne Particulate Sampling in Horse Barns

Measurements of total, respirable, and real-time airborne particulate were obtained in 12 horse barns in summer and winter. Respirable and total particulate concentrations were measured gravimetrically; real-time particulate was measured with an aerosol photometer. Total particulate (TP) ranged from nondetectable (ND) to 2.1 mg/m3 and from ND to 1.2 mg/m3 for winter and summer sampling, respectively. Respirable particulate (RP) ranged from ND to 0.2 mg/m3 and from ND to 0.7 mg/m3 for winter and summer measurements, respectively. The mean respirable fraction of particulate for summer and winter measurements was 0.63 and 0.34, respectively Real-time particulate (RTP) concentration was significantly correlated with TP for winter measurements and for pooled data but was not significantly correlated with RP for winter, summer, or pooled measurements. Peak, 10-sec average levels of RTP were estimated to range up to 22 mg/m3 in the samples collected. The data suggest that (a) airborne particulate concentrations and particle size distributions vary considerably among horse barns, and (b) real-time sampling, with an aerosol photometer, may be a more reliable measure of airborne TP than airborne RP in these environments.

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%–3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%–2.5% of the target level from years 1–5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated.

Progress with Recombinant Adeno-Associated Virus Vectors for Gene Therapy of Alpha-1 Antitrypsin Deficiency

The pathway to a clinical gene therapy product often involves many changes of course and strategy before obtaining successful results. Here we outline the methodologies, both clinical and preclinical, that went into developing a gene therapy approach to the treatment of alpha-1 antitrypsin deficiency lung disease using muscle-targeted recombinant adeno-associated virus. From initial gene construct development in mouse models through multiple rounds of safety and biodistribution studies in rodents, rabbits, and nonhuman primates to ultimate human trials, this review seeks to provide insight into what clinical translation entails and could thereby inform the process for future investigators.

Stability and Compatibility of Recombinant Adeno-Associated Virus Under Conditions Commonly Encountered in Human Gene Therapy Trials

Recombinant adeno-associated virus (rAAV) vectors are rapidly becoming the first choice for human gene therapy studies, as clinical efficacy has been demonstrated in several human trials and proof-of-concept data have been demonstrated for correction of many others. When moving into human use under the auspices of an FDA Investigational New Drug (IND) application, it is necessary to demonstrate the stability of vector material under various conditions of storage, dilution, and administration when used in humans. Limited data are currently available in the literature regarding vector compatibility and stability, leading most IND sponsors to repeat all necessary studies. The current study addresses this issue with an rAAV vector (rAAV1-CB-chAATmyc) containing AAV2-inverted terminal repeat sequences packaged into an AAV1 capsid. Aliquots of vector were exposed to a variety of temperatures, diluents, container constituents, and other environmental conditions, and its functional biological activity (after these various treatments) was assessed by measuring transgene expression after intramuscular injection in mice. rAAV was found to be remarkably stable at temperatures ranging from 4°C to 55°C (with only partial loss of potency after 20 min at 70°C), at pH ranging from 5.5 to 8.5, after contact with mouse or human serum (with or without complement depletion) or with gadolinium and after contact with glass, polystyrene, polyethylene, polypropylene, and stainless steel. The only exposure resulting in near-total loss of vector activity (10,000-fold loss) was UV exposure for 10 min. The stability of rAAV1 preparations bodes well for future dissemination of this therapeutic modality.