Nanomedicine has been widely employed as a site‐specific and target‐oriented drug delivery tactic in treating heart failure (HF). Ischemia‐induced inflammation plays a robust role in the development of HF and neutrophil is one of the main immune cell types that rapidly infiltrate into the inflamed tissues after myocardial infarction (MI). In the present study, we examined the efficacy of a formation of biomimetic nanoparticles that were prepared by fabricated TAPI‐1 loaded liposomes coated with neutrophil membranes in the treatment of HF. Male SD rats underwent coronary artery ligation (CL) to induce HF. These HF rats were treated with intravenous injection of TAPI‐1‐loaded biomimetic nanoparticles (n=6) or TAPI‐1 alone (n=6) for 4 weeks. The left ventricular (LV) function was assessed by echocardiography within 24 hours and 4 weeks after CL. The nanoparticles were evaluated prior to the treatments. Compared with non‐coated formation, neutrophil membrane‐coated nanoparticles have high uptake into LPS‐stimulated cardiac cells or endothelial cells. Moreover, the coated nanoparticles exhibited higher cell‐specific targeting potential and internalization capacity than non‐coated nanoparticles in LPS‐stimulated endothelial cells. Small animal imaging showed that DiR‐labeled nanoparticles were mainly accumulated in the injured heart after MI. Compared with HF rats treated with TAPI‐1 alone, the HF rats treated with TAPI‐1‐loaded nanoparticles had significantly (* p<0.05) improved anatomic indicators with reduced ratio (%) of heart/body weight (0.34 ± 0.042* vs. 0.48± 0.11) and lung/body weight (0.84 ± 0.19* vs. 1.14 ± 0.15). Additionally, one 4‐week treatment with TAPI‐1‐loaded biomimetic nanoparticles reduced LV end systolic volume (0.67±0.032* vs. 0.83±0.036, mL) when compared with the TAPI‐1 alone. While LV stroke volume (LVSV) did not change significantly in IV TAPI‐1‐treated HF animals, LVSV (0.47±0.043* vs. 0.27±0.029, mL) was significantly increased in HF rats treated with TAPI‐1‐loaded nanoparticles at 4 weeks vs. 24 hours after CL. LV end diastolic volume, LV ejection fraction, and LV infarction zone didn’t exhibit significant differences between two treatment groups. Cardiac hemodynamic measurements revealed that the HF rats treated with TAPI‐1‐loaded nanoparticles had decreased LV end diastolic pressure (10.53±1.67* vs. 19.01±3.32, mmHg) and elevated dP/dtmax(8202.65±658.09* vs. 5994.47±630.81) compared with the HF rats‐treated with TAPI‐1 alone. Histological evaluation indicated that the fibrosis areas surrounding the infarction scar were significant reduced (15.97±1.84* vs. 22.86±2.27, %) in HF rats treated with TAPI‐1‐loaded nanoparticles vs. TAPI alone. Flow cytometric analysis also demonstrated that treatment with TAPI‐1‐loaded nanoparticles improved inflammatory conditions indicated by increased ratio of CD4+/CD8+ T Cells (2.40±0.67* vs. 1.56±0.21) in the blood and reduced percentage of leukocytes in the heart of HF. These data indicate that neutrophil membrane fusogenic liposomal nanoparticle is an effective drug delivery approach in treating this devastating disease. (Supported by NIH grants R01 HL‐139521 & HL‐155091 to SGW, S10 OD019941 to RW)

Spleen plays an important role in promoting inflammation in myocardial infarction (MI)‐induced heart failure (HF). Spleen may have the temporal and/or permanent changes to accommodate the neurohumoral compensatory responses in the context of HF. However, the mechanisms underlying the immune cell deployment from the spleen in HF remain elusive. To gain unbiased insights into how splenic cells are transcriptionally adapted to the neurohumoral alterations in HF, we performed RNA‐seq analysis on spleen‐derived leukocytes from HF rats or sham‐operated (Sham) rats. Male Sprague‐Dawley rats underwent coronary artery ligation to induce HF (n=3) or Sham (n=4). HF was confirmed by echocardiography. Splenic leukocytes were prepared two weeks after MI. RNA were extracted, sequenced and analyzed with standard RNA sequencing procedure. We found 466 upregulated and 113 downregulated genes in splenic leukocytes of HF rats (|log2FoldChanges|>0.2, p<0.05). The genes associated with erythropoiesis (e.g Trim10, Dmtn, and Spta1) and cell cycles regulatory factors such as Ki‐67, and polo‐like kinase2 (plk2) were transcriptionally activated and the enhanced expression was validated using RT‐qPCR, indicating that active myeloid proliferation and differentiation are present within the spleen in the development of HF. Unexpectedly, the expression of inflammation‐related genes such as chemokine ccl3, JUN, JUNB, and fosB were reduced in HF. Moreover, pathway enrichment analysis also displayed a suppression of the immune‐related pathways including cytokine‐cytokine interaction pathway, IL‐17 signaling, tumor necrosis factor signaling, and rheumatoid arthritis. To determine whether the suppression of these pathways is resulted from the egress of immune cells in spleen, we performed flow cytometry profiling of splenic cells. We found that CD3+T cell (21% vs. 27%*), especially CD8+ T cells (7.5 % vs 12%*) were significantly (*p<0.05) reduced in HF compared with Sham animals. However, the immune cells in other categories such as monocytes (M1, CD43low;HIS48high: 3% vs. 2%*; M2, CD43high;HIS48low: 5% vs. 3%*), NK cells (5% vs. 4%*), and neutrophiles (2.5% vs. 1%*) were significantly elevated in HF. The B cells remain unchanged. Flow cytometrical analysis of immune cell subtypes in the blood of HF showed a similar trend with those of splenic leukocytes. We proposed that the sympathetic activation in HF may contribute to the dynamic changes of splenocytes. To test this hypothesis, norepinephrine (NE) was injected (0.1mg/kg, IP) in normal rats (n=4) for 3 consecutive days followed by flow cytometry analysis of splenic leukocytes. The results indicated that, while all other immune cell subtypes remain unchanged, both M1 (2% vs. 1.4%*) and M2 (5% vs. 2%*) monocytes are decreased in NE‐treated rats, implying that elevated NE level at early stage of HF can robustly trigger the infiltration of the spleen‐derived monocytes into injured cardiac tissues. Taken together, these data provided an informative perspective to understand splenic modulations in HF. The dynamic changes of splenocytes composition in HF is most likely a combined result of splenic cell deployment and compensatory proliferation/differentiation. Our data also suggest that the sympathetic overactivity may play an important role in the migration of spleen‐resident leukocytes and inflammatory regulation in HF.

Tumor necrosis factor (TNF)‐α converting enzyme (TACE), also known as a disintegrin and metalloprotease (ADAM)17, is a key mediator of cell signaling by proteolytically cleaving extracellular domains of various cytokines and growth factors. TACE‐mediated shedding of transforming growth factor (TGF)‐α, has been shown to transactivate epidermal growth factor receptor (EGFR) to activate the mitogen‐activated protein kinase signaling pathway in the pathophysiological conditions. We previously reported that both TACE and TGF‐α are upregulated in the hypothalamic paraventricular nucleus (PVN, a critical cardiovascular and autonomic center) and contribute to the sympathetic excitation in heart failure (HF). However, the mechanisms by which TACE and TGF‐α are upregulated in the PVN in HF remain unclear. Recent evidence reveals that inactive Rhomboid proteins (iRhoms) including iRhom1 and iRhom2 are essential upstream regulators of TACE/ADAM17. The present study sought to determine the expression of iRhoms and its relationship with TACE‐mediated shedding of TGF‐α in the PVN in HF. Male rats underwent coronary artery ligation to induce HF or sham surgery (Sham). The left ventricular function and infarction size were assessed by echocardiography. Four weeks later, these animals were euthanized to collect cerebrospinal fluid (CSF), PVN tissues and blood for molecular and biochemical measurements. All values are expressed as the mean ± SD. Real‐time PCR showed abundant mRNA expression of both iRhom1 and iRhom2 in the PVN and brain cortex in Sham or HF rats. Compared with Sham rats, HF rats had significantly (*P<0.05) increased mRNA expression of iRhom2 (2.00 ± 0.57* vs 1.03 ± 0.28, HF vs Sham) but not iRhom1 (1.32 ± 0.41 vs 1.02 ± 0.21) in the PVN. Additionally, HF rats exhibited marked increases in mRNA expression of TACE (2.25 ± 1.15* vs 1.04 ± 0.36) in the PVN and the plasma level of norepinephrine (NE, a marker of sympathetic excitation, 12.39 ± 4.96* vs 5.82 ± 2.76 ng/mL), along with elevated levels of TGF‐α (23.54 ± 6.25* vs 10.08 ± 4.38 pg/mL) in CSF. The levels of epidermal growth factor (9.88 ± 3.59 vs 8.15 ± 4.24 pg/mL) in the CSF did not alter significantly in HF compared with Sham rats. Moreover, the linear regression analysis indicated that the mRNA expression of iRhom2 in the PVN was positively correlated with TACE level in the PVN (R2 = 0.62, P <0.01), TGF‐α level in the CSF (R2 = 0.64, P <0.01) and NE level in the plasma (R2 = 0.63, P <0.01). These results suggest that the upregulated expression of iRhom2 in the PVN may promote ectodomain shedding of TGF‐α by enhancing TACE expression to drive sympathetic activation in HF. Brain iRhom2 is a potential target for therapeutic intervention in HF.

TACE is a key metalloprotease involved in ectodomain shedding of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-α. We previously reported that TACE-mediated production of TNF-α in the hypothalamic paraventricular nucleus (PVN) contributes to the sympathetic excitation in heart failure (HF). Additionally, the upregulated TGF-α in the PVN transactivates the epidermal growth factor receptor (EGFR) to activate extracellular signal-regulated kinase (ERK) 1/2 in HF. Here we sought to determine whether central inhibition of TACE attenuates neuroinflammation and prevents the progress of HF. Male rats underwent coronary artery ligation to induce HF or sham surgery (Sham). These rats were treated with bilateral PVN microinjection of a TACE siRNA or control siRNA while some rats received a 4-week intracerebroventricular (ICV) infusion of TACE inhibitor TAPI-0 or vehicle. Compared with Sham rats, HF rats treated with control siRNA, had higher (*P<0.05) levels of TNF-α (7.88±1.32* vs 2.77±0.98 pg/mL) and TGF-α (28.27±2.76* vs 11.62±2.48 pg/mL) in cerebrospinal fluid, and increased mRNA expression of TACE (2.53±0.30* vs 1.04±0.12), TNF-α (3.43±0.55* vs 1.03±0.11), TNF-α receptor 1 (2.32±0.27* vs 1.07±0.19), cyclooxygenase-2 (2.96±0.31* vs 1.10±0.19) and TGF-α (2.68±0.41* vs 1.06±0.14) in the PVN, but these levels were markedly reduced (39-54%*) in TACE siRNA-treated HF rats. Compared with control HF rats, HF rats treated with TACE siRNA had reduced expression of phosphorylated (p-) NF-κB p65 (1.27±0.14 vs 0.84±0.07*), p-EGFR (0.52±0.05 vs 0.37±0.04*) and p-ERK1/2 (1.06±0.10 vs 0.62±0.09*) in the PVN. Moreover, the elevated plasma norepinephrine levels, lung/body weight, heart/body weight and left ventricular (LV) end-diastolic pressure along with decreased LV dP/dt max in HF rats-treated with control siRNA were significantly attenuated in HF rats treated with TACE siRNA. Treatments with TACE siRNA in the PVN also improved the indicators of cardiac hypertrophy and fibrosis of HF. ICV infusion of TAPI-0 had the similar effects with PVN TACE siRNA on these variables in HF. These data indicate that central interventions suppressing TACE activity ameliorate neuroinflammation, sympathetic activation and cardiac dysfunction in HF.