Oral Delivery of Angiotensin-Converting Enzyme 2 and Angiotensin-(1-7) Bioencapsulated in Plant Cells Attenuates Pulmonary Hypertension

Emerging evidences indicate that diminished activity of the vasoprotective axis of the renin–angiotensin system, constituting angiotensin-converting enzyme 2 (ACE2) and its enzymatic product, angiotensin-(1-7) [Ang-(1-7)] contribute to the pathogenesis of pulmonary hypertension (PH). However, long-term repetitive delivery of ACE2 or Ang-(1-7) would require enhanced protein stability and ease of administration to improve patient compliance. Chloroplast expression of therapeutic proteins enables their bioencapsulation within plant cells to protect against gastric enzymatic degradation and facilitates long-term storage at room temperature. Besides, fusion to a transmucosal carrier helps effective systemic absorption from the intestine on oral delivery. We hypothesized that bioencapsulating ACE2 or Ang-(1-7) fused to the cholera nontoxin B subunit would enable development of an oral delivery system that is effective in treating PH. PH was induced in male Sprague Dawley rats by monocrotaline administration. Subset of animals was simultaneously treated with bioencapsulaed ACE2 or Ang-(1-7) (prevention protocol). In a separate set of experiments, drug treatment was initiated after 2 weeks of PH induction (reversal protocol). Oral feeding of rats with bioencapsulated ACE2 or Ang-(1-7) prevented the development of monocrotaline-induced PH and improved associated cardiopulmonary pathophysiology. Furthermore, in the reversal protocol, oral ACE2 or Ang-(1-7) treatment significantly arrested disease progression, along with improvement in right heart function, and decrease in pulmonary vessel wall thickness. In addition, a combination therapy with ACE2 and Ang-(1-7) augmented the beneficial effects against monocrotaline-induced lung injury. Our study provides proof-of-concept for a novel low-cost oral ACE2 or Ang-(1-7) delivery system using transplastomic technology for pulmonary disease therapeutics

Oral Delivery of Angiotensin-Converting Enzyme 2 and Angiotensin-(1-7) Bioencapsulated in Plant Cells Attenuates Pulmonary Hypertension

Emerging evidences indicate that diminished activity of the vasoprotective axis of the renin–angiotensin system, constituting angiotensin-converting enzyme 2 (ACE2) and its enzymatic product, angiotensin-(1-7) [Ang-(1-7)] contribute to the pathogenesis of pulmonary hypertension (PH). However, long-term repetitive delivery of ACE2 or Ang-(1-7) would require enhanced protein stability and ease of administration to improve patient compliance. Chloroplast expression of therapeutic proteins enables their bioencapsulation within plant cells to protect against gastric enzymatic degradation and facilitates long-term storage at room temperature. Besides, fusion to a transmucosal carrier helps effective systemic absorption from the intestine on oral delivery. We hypothesized that bioencapsulating ACE2 or Ang-(1-7) fused to the cholera nontoxin B subunit would enable development of an oral delivery system that is effective in treating PH. PH was induced in male Sprague Dawley rats by monocrotaline administration. Subset of animals was simultaneously treated with bioencapsulaed ACE2 or Ang-(1-7) (prevention protocol). In a separate set of experiments, drug treatment was initiated after 2 weeks of PH induction (reversal protocol). Oral feeding of rats with bioencapsulated ACE2 or Ang-(1-7) prevented the development of monocrotaline-induced PH and improved associated cardiopulmonary pathophysiology. Furthermore, in the reversal protocol, oral ACE2 or Ang-(1-7) treatment significantly arrested disease progression, along with improvement in right heart function, and decrease in pulmonary vessel wall thickness. In addition, a combination therapy with ACE2 and Ang-(1-7) augmented the beneficial effects against monocrotaline-induced lung injury. Our study provides proof-of-concept for a novel low-cost oral ACE2 or Ang-(1-7) delivery system using transplastomic technology for pulmonary disease therapeutics

KCNK3 mutation causes altered immune function in pulmonary arterial hypertension patients and mouse models

Loss of function KCNK3 mutation is one of the gene variants driving hereditary pulmonary arterial hypertension (PAH). KCNK3 is expressed in several cell and tissue types on both membrane and endoplasmic reticulum and potentially plays a role in multiple pathological process associated with PAH. However, the role of various stressors driving the susceptibility of KCNK3 mutation to PAH is unknown. Hence, we expose

Complementary Embryonic and Adult Cell Populations Enhance Myocardial Repair in Rat Myocardial Injury Model

We compared the functional outcome of Isl-1+ cardiac progenitors, CD90+ bone marrow-derived progenitor cells, and the combination of the two in a rat myocardial infarction (MI) model. Isl-1+ cells were isolated from embryonic day 12.5 (E12.5) rat hearts and expanded in vitro. Thy-1+/CD90+ cells were isolated from the bone marrow of adult Sprague-Dawley rats by immunomagnetic cell sorting. Six-week-old female Sprague-Dawley rats underwent permanent left anterior descending (LAD) coronary artery ligation and received intramyocardial injection of either saline, Isl-1+ cells, CD90+ cells, or a combination of Isl-1+ and CD90+ cells, at the time of infarction. Cells were delivered transepicardially to the peri-infarct zone. Left ventricular function was assessed by transthoracic echocardiography at 1- and 4-week post-MI and by Millar catheterization (-dP/dt and +dP/dt) at 4-week post-MI. Fluorescence in situ hybridization (Isl-1+cells) and monochrystalline iron oxide nanoparticles labeling (MION; CD90+ cells) were performed to assess biodistribution of transplanted cells. Only the combination of cells demonstrated a significant improvement of cardiac function as assessed by anterior wall contractility, dP/dt (max), and dP/dt (min), compared to Isl-1+ or CD90+ cell monotherapies. In the combination cell group, viable cells were detected at week 4 when anterior wall motion was completely restored. In conclusion, the combination of Isl-1+ cardiac progenitors and adult bone marrow-derived CD90+ cells shows prolonged and robust myocardial tissue repair and provides support for the use of complementary cell populations to enhance myocardial repair

Overexpression of Msx1 in Mouse Lung Leads to Loss of Pulmonary Vessels Following Vascular Hypoxic Injury

Pulmonary arterial hypertension (PAH) is a progressive lung disease caused by thickening of the pulmonary arterial wall and luminal obliteration of the small peripheral arteries leading to increase in vascular resistance which elevates pulmonary artery pressure that eventually causes right heart failure and death. We have previously shown that transcription factor Msx1 (mainly expressed during embryogenesis) is strongly upregulated in transformed lymphocytes obtained from PAH patients, especially IPAH. Under pathological conditions, Msx1 overexpression can cause cell dedifferentiation or cell apoptosis. We hypothesized that Msx1 overexpression contributes to loss of small pulmonary vessels in PAH. In IPAH lung, MSX1 protein localization was strikingly increased in muscularized remodeled pulmonary vessels, whereas it was undetectable in control pulmonary arteries. We developed a transgenic mouse model overexpressing MSX1 (MSX1OE) by about 4-fold and exposed these mice to normoxic, sugen hypoxic (3 weeks) or hyperoxic (100% 02 for 3 weeks) conditions. Under normoxic conditions, compared to controls, MSX1OE mice demonstrated a 30-fold and 2-fold increase in lung Msx1 mRNA and protein expression, respectively. There was a significant retinal capillary dropout (p < 0.01) in MSX1OE mice, which was increased further (p < 0.03) with sugen hypoxia. At baseline, the number of pulmonary vessels in MSX1OE mice was similar to controls. In sugen-hypoxia-treated MSX1OE mice, the number of small (0–25 uM) and medium (25–50 uM) size muscularized vessels increased approximately 2-fold (p < 0.01) compared to baseline controls; however, they were strikingly lower (p < 0.001) in number than in sugen-hypoxia-treated control mice. In MSX1OE mouse lung, 104 genes were upregulated and 67 genes were downregulated compared to controls. Similarly, in PVECs, 156 genes were upregulated and 320 genes were downregulated from siRNA to MSX1OE, and in PVSMCs, 65 genes were upregulated and 321 genes were downregulated from siRNA to MSX1OE (with control in the middle). Many of the statistically significant GO groups associated with MSX1 expression in lung, PVECs, and PVSMCs were similar, and were involved in cell cycle, cytoskeletal and macromolecule organization, and programmed cell death. Overexpression of MSX1 suppresses many cell-cycle-related genes in PVSMCs but induces them in PVECs. In conclusion, overexpression of Msx1 leads to loss of pulmonary vessels, which is exacerbated by sugen hypoxia, and functional consequences of Msx1 overexpression are cell-dependent

The Selective Angiotensin II Type 2 Receptor Agonist, Compound 21, Attenuates the Progression of Lung Fibrosis and Pulmonary Hypertension in an Experimental Model of Bleomycin-Induced Lung Injury

Idiopathic Pulmonary Fibrosis (IPF) is a chronic lung disease characterized by scar formation and respiratory insufficiency, which progressively leads to death. Pulmonary hypertension (PH) is a common complication of IPF that negatively impacts clinical outcomes, and has been classified as Group III PH. Despite scientific advances, the dismal prognosis of IPF and associated PH remains unchanged, necessitating the search for novel therapeutic strategies. Accumulating evidence suggests that stimulation of the angiotensin II type 2 (AT2) receptor confers protection against a host of diseases. In this study, we investigated the therapeutic potential of Compound 21 (C21), a selective AT2 receptor agonist in the bleomycin model of lung injury. A single intra-tracheal administration of bleomycin (2.5 mg/kg) to 8-week old male Sprague Dawley rats resulted in lung fibrosis and PH. Two experimental protocols were followed: C21 was administered (0.03 mg/kg/day, ip) either immediately (prevention protocol, BCP) or after 3 days (treatment protocol, BCT) of bleomycin-instillation. Echocardiography, hemodynamic, and Fulton's index assessments were performed after 2 weeks of bleomycin-instillation. Lung tissue was processed for gene expression, hydroxyproline content (a marker of collagen deposition), and histological analysis. C21 treatment prevented as well as attenuated the progression of lung fibrosis, and accompanying PH. The beneficial effects of C21 were associated with decreased infiltration of macrophages in the lungs, reduced lung inflammation and diminished pulmonary collagen accumulation. Further, C21 treatment also improved pulmonary pressure, reduced muscularization of the pulmonary vessels and normalized cardiac function in both the experimental protocols. However, there were no major differences in any of the outcomes measured from the two experimental protocols. Collectively, our findings indicate that stimulation of the AT2 receptor by C21 attenuates bleomycin-induced lung injury and associated cardiopulmonary pathology, which needs to be further explored as a promising approach for the clinical treatment of IPF and Group III PH

Pulmonary veno-occlusive disease in Sjogren's syndrome: a case report

Abstract Background Pulmonary arterial hypertension (PAH) associated with connective tissue disease (CTD) belongs to Group 1 pulmonary hypertension. Pulmonary veno-occlusive disease (PVOD), which is characterized by venous system aberrations, has been previously reported in CTD-PAH; however, it has rarely been observed in Sjogren’s syndrome (SS). Case presentation Our 28-year-old female patient was admitted to the hospital with recurrent shortness of breath even after minimal physical activity. Her chest high-resolution CT scan demonstrated pulmonary artery dilatation and bilateral ground-glass nodules. A subsequent right heart catheterization confirmed pulmonary hypertension because her mean pulmonary arterial pressure was 62 mmHg. Our inquisitive genomic assessment identified a novel EIF2AK4 mutation at c.1021 C > T (p. Gln341*), the dominant causal gene of PVOD. Histological examination demonstrated stenosis and occlusions in the pulmonary veins. Because she presented with features such as dry eyes and Raynaud's phenomenon, we performed a biopsy on the labial salivary gland, which confirmed SS. Her treatment regimen included PAH-targeted therapies (tadalafil and macitentan) in combination with hydroxychloroquine. Although she was hospitalized several times due to acute exacerbation of PAH, her disease progression was under control, and she did not demonstrate any signs of pulmonary edema even after a three-year treatment period. Conclusion Here, we report the case of an SS-PAH patient with PVOD who carried a novel biallelic EIF2AK4 mutation, and PAH-targeted therapies were well tolerated by our patient

rhACE2 Therapy Modifies Bleomycin-Induced Pulmonary Hypertension via Rescue of Vascular Remodeling

Background: Pulmonary hypertension (PH) is a progressive cardiovascular disease, characterized by endothelial and smooth muscle dysfunction and vascular remodeling, followed by right heart failure. Group III PH develops secondarily to chronic lung disease such as idiopathic pulmonary fibrosis (IPF), and both hastens and predicts mortality despite of all known pharmacological interventions. Thus, there is urgent need for development of newer treatment strategies.Objective: Angiotensin converting enzyme 2 (ACE2), a member of the renin angiotensin family, is therapeutically beneficial in animal models of pulmonary vascular diseases and is currently in human clinical trials for primary PH. Although previous studies suggest that administration of ACE2 prevents PH secondary to bleomycin-induced murine IPF, it is unknown whether ACE2 can reverse or treat existing disease. Therefore, in the present study, we tested the efficacy of ACE2 in arresting the progression of group 3 PH.Methods: To establish pulmonary fibrosis, we administered 0.018 U/g bleomycin 2x/week for 4 weeks in adult FVB/N mice, and sacrificed 5 weeks following the first injection. ACE2 or vehicle was administered via osmotic pump for the final 2 weeks, beginning 3 weeks after bleomycin. Echocardiography and hemodynamic assessment was performed prior to sacrifice and tissue collection.Results: Administration of bleomycin significantly increased lung collagen expression, pulmonary vascular remodeling, and pulmonary arterial pressure, and led to mild right ventricular hypertrophy. Acute treatment with ACE2 significantly attenuated vascular remodeling and increased pulmonary SOD2 expression without measurable effects on pulmonary fibrosis. This was associated with nonsignificant positive effects on pulmonary arterial pressure and cardiac function.Conclusion: Collectively, our findings enumerate that ACE2 treatment improved pulmonary vascular muscularization following bleomycin exposure, concomitant with increased SOD2 expression. Although it may not alter the pulmonary disease course of IPF, ACE2 could be an effective therapeutic strategy for the treatment of group 3 PH