Elizabeth R. Flynn

Omega-3 fatty acid rich diet prevents diabetic renal disease

Omega-3 polyunsaturated fatty acids (n-3 PUFA) show beneficial effects in cardiovascular disease, IgA, and diabetic nephropathy; however, the mechanisms underlying these benefits are unknown. The study was performed in male Sprague-Dawley rats randomly divided into four treatment groups: nondiabetic (ND), streptozotocin-induced diabetic (D), diabetic and fed a high n-3 PUFA diet (D+canola), and diabetic and fed a high n-6 (omega-6) PUFA diet (D+corn). Study treatments were carried out for 30 wk. D+canola significantly decreased diabetes-associated increases in urine albumin excretion (ND 17.8 +/- 6.4; D 97.3 +/- 9.4; D+canola 8.3 +/- 2.2 mg/day); systolic blood pressure (ND 153 +/- 9; D 198 +/- 7; D+canola 162 +/- 9 mmHg); glomerulosclerosis (ND 0.6 +/- 0.2; D 1.8 +/- 0.2; D+canola 0.8 +/- 0.1 AU); and tubulointerstitial fibrosis in the renal cortex (ND 1.2 +/- 0.2; D 2.0 +/- 0.2; D+canola 1.1 +/- 0.1) and the inner stripe of the outer medulla (ND 1.0 +/- 0.2; D 2.1 +/- 0.2; D+canola 1.1 +/- 0.2 AU). D+corn also exerted renoprotection, but not to the same degree as D+canola (urine albumin excretion, 33.8 +/- 6.1 mg/day; systolic blood pressure, D+corn 177 +/- 6 mmHg; glomerulosclerosis, D+corn 1.2 +/- 0.3 AU; cortical tubulointerstitial fibrosis, D+corn 1.6 +/- 0.1 AU; medullary tubulointerstitial fibrosis, D+corn 1.5 +/- 0.1 AU). In addition, D+canola attenuated D-associated increase in collagen type I and type IV, IL-6, MCP-1, transforming growth factor-beta, and CD68 expression. These observations indicate a beneficial effect of high dietary intake of n-3 PUFA in reducing diabetic renal disease.

A Novel Collagen Matricryptin Reduces Left Ventricular Dilation Post-Myocardial Infarction by Promoting Scar Formation and Angiogenesis

BACKGROUND Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). OBJECTIVES This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). METHODS Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. RESULTS In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mouse LV, at levels that inversely correlated to MMP-9 levels. We synthesized a peptide beginning at the cleavage site (p1158/59, amino acids 1159 to 1173) to investigate its biological functions. In vitro, p1158/59 stimulated fibroblast wound healing and robustly promoted angiogenesis. In vivo, early post-MI treatment with p1158/59 reduced LV dilation at day 7 post-MI by preserving LV structure (p < 0.05 vs. control). The p1158/59 stimulated both in vitro and in vivo wound healing by enhancing basement membrane proteins, granulation tissue components, and angiogenic factors. CONCLUSIONS Collagen Iα1 matricryptin p1158/59 facilitates LV remodeling post-MI by regulating scar formation through targeted ECM generation and stimulation of angiogenesis.

Temporal Neutrophil Polarization Following Myocardial Infarction

AIMS Although macrophage phenotypes have been well studied in the myocardial infarction (MI) setting, this study investigated temporal neutrophil polarization and activation mechanisms. METHODS AND RESULTS Neutrophils isolated from the infarcted left ventricle (LV) of mice showed high expression of proinflammatory markers at Day 1 and anti-inflammatory markers at Days 5 and 7 post-MI, indicating distinct neutrophil phenotypes along the post-MI time continuum. Flow cytometry analysis revealed that although proinflammatory N1 neutrophils were always predominant (>80% of total neutrophils at each time point), the percentage of N2 neutrophils increased post-MI from 2.4 ± 0.6% at Day 1 to 18.1 ± 3.0% at Day 7. In vitro, peripheral blood neutrophils were polarized to proinflammatory N1 by lipopolysaccharide and interferon-γ or anti-inflammatory N2 by interleukin-4, indicating high plasticity potential. The in vivo post-MI relevant LV damage-associated molecular patterns (DAMPs) polarized neutrophils to a proinflammatory N1 phenotype by activating toll-like receptor-4. Transforming growth factor-β1 inhibited proinflammatory production in neutrophils. N1 neutrophils positively correlated with infarct wall thinning at Day 7 post-MI, possibly due to high production of matrix metalloproteinases-12 and -25. CONCLUSION This study is the first to identify the existence of N1 and N2 neutrophils in the infarct region and reveals that N1 polarization could be mediated by DAMPs.

Deriving a cardiac ageing signature to reveal MMP-9-dependent inflammatory signalling in senescence

AIMS Cardiac ageing involves the progressive development of cardiac fibrosis and diastolic dysfunction coordinated by MMP-9. Here, we report a cardiac ageing signature that encompasses macrophage pro-inflammatory signalling in the left ventricle (LV) and distinguishes biological from chronological ageing. METHODS AND RESULTS Young (6-9 months), middle-aged (12-15 months), old (18-24 months), and senescent (26-34 months) mice of both C57BL/6J wild type (WT) and MMP-9 null were evaluated. Using an identified inflammatory pattern, we were able to define individual mice based on their biological, rather than chronological, age. Bcl6, Ccl24, and Il4 were the strongest inflammatory markers of the cardiac ageing signature. The decline in early-to-late LV filling ratio was most strongly predicted by Bcl6, Il1r1, Ccl24, Crp, and Cxcl13 patterns, whereas LV wall thickness was most predicted by Abcf1, Tollip, Scye1, and Mif patterns. With age, there was a linear increase in cardiac M1 macrophages and a decrease in cardiac M2 macrophages in WT mice; of which, both were prevented by MMP-9 deletion. In vitro, MMP-9 directly activated young macrophage polarization to an M1/M2 mid-transition state. CONCLUSION Our results define the cardiac ageing inflammatory signature and assign MMP-9 roles in mediating the inflammaging profile by indirectly and directly modifying macrophage polarization. Our results explain early mechanisms that stimulate ageing-induced cardiac fibrosis and diastolic dysfunction.

Microvascular disease precedes the decline in renal function in the streptozotocin-induced diabetic rat

Diabetic nephropathy is a progressive and generalized vasculopathic condition associated with abnormal angiogenesis. We aim to determine whether changes in renal microvascular (MV) density correlate with and play a role in the progressive deterioration of renal function in diabetes. We hypothesize that MV changes represent the early steps of renal injury that worsen as diabetes progresses, initiating a vicious circle that leads to irreversible renal injury. Male nondiabetic (ND) or streptozotocin-induced diabetic (D) Sprague-Dawley rats were followed for 4 or 12 wk. Renal blood flow and glomerular filtration rate (GFR) were measured by PAH and (125)I-[iothalamate], respectively. Renal MV density was quantified ex vivo using three-dimensional micro computed tomography and JG-12 immunoreactivity. Vascular endothelial growth factor (VEGF) levels (ELISA) and expression of VEGF receptors and factors involved in MV remodeling were quantified in renal tissue by Western blotting. Finally, renal morphology was investigated by histology. Four weeks of diabetes was associated with increased GFR, accompanied by a 34% reduction in renal MV density and augmented renal VEGF levels. However, at 12 wk, while GFR remained similarly elevated, reduction of MV density was more pronounced (75%) and associated with increased MV remodeling, renal fibrosis, but unchanged renal VEGF compared with ND at 12 wk. The damage, loss, and subsequent remodeling of the renal MV architecture in the diabetic kidney may represent the initiating events of progressive renal injury. This study suggests a novel concept of MV disease as an early instigator of diabetic kidney disease that may precede and likely promote the decline in renal function.

Preventing Autoimmunity Protects Against the Development of Hypertension and Renal Injury

Several studies suggest a link between autoimmunity and essential hypertension in humans. However, whether autoimmunity can drive the development of hypertension remains unclear. The autoimmune disease systemic lupus erythematosus is characterized by autoantibody production, and the prevalence of hypertension is increased markedly in this patient population compared with normal healthy women. We hypothesized that preventing the development of autoimmunity would prevent the development of hypertension in a mouse model of lupus. Female lupus (NZBWF1) and control mice (NZW) were treated weekly with anti-CD20 or immunoglobulin G antibodies (both 10 mg/kg, IV) starting at 20 weeks of age for 14 weeks. Anti-CD20 therapy markedly attenuated lupus disease progression as evidenced by reduced CD45R+ B cells and lower double-stranded DNA autoantibody activity. In addition, renal injury in the form of urinary albumin, glomerulosclerosis, and tubulointerstitial fibrosis, as well as tubular injury (indicated by renal cortical expression of neutrophil gelatinase-associated lipocalin) was prevented by anti-CD20 therapy in lupus mice. Finally, lupus mice treated with anti-CD20 antibody did not develop hypertension. The protection against the development of hypertension was associated with lower renal cortical tumor necrosis factor-&agr; expression, a cytokine that has been previously reported by us to contribute to the hypertension in this model, as well as renal cortical monocyte chemoattractant protein-1 expression and circulating T cells. These data suggest that the development of autoimmunity and the resultant increase in renal inflammation are an important underlying factor in the prevalent hypertension that occurs during systemic lupus erythematosus.

Exposure to maternal overnutrition and a high-fat diet during early postnatal development increases susceptibility to renal and metabolic injury later in life

Overnutrition during pre- and postnatal development both confer increased susceptibility to renal and metabolic risks later in life; however, whether they have an additive effect on the severity of renal and metabolic injury remains unknown. The present study tested the hypothesis that a combination of a pre- and postnatal diet high in fat/fructose would exacerbate renal and metabolic injury in male offspring later in life. Male offspring born to high fat/high-fructose-fed mothers and fed a high-fat/high-fructose diet postnatally (HF-HF) had increased urine albumin excretion (450%), glomerulosclerosis (190%), and tubulointerstitial fibrosis (101%) compared with offspring born to mothers fed a standard diet and fed a standard diet postnatally (NF-NF). No changes in blood pressure or glomerular filtration were observed between any of the treatment groups. The HF-HF offspring weighed ∼23% more than offspring born to mothers fed a high-fat/high-fructose diet and fed a normal diet postnatally (HF-NF), as well as offspring born to mothers fed a standard diet regardless of their postnatal diet. The HF-HF rats also had increased (and more variable) blood glucose levels over 12 wk of being fed a high-fat/high-fructose diet. A combination of exposure to a high-fat/high-fructose diet in utero and postnatally increased plasma insulin levels by 140% compared with NF-NF offspring. Our data suggest that the combined exposure to overnutrition during fetal development and early postnatal development potentiate the susceptibility to renal and metabolic disturbances later in life.

Building a better infarct: Modulation of collagen cross-linking to increase infarct stiffness and reduce left ventricular dilation post-myocardial infarction

Matrix metalloproteinase-9 (MMP-9) deletion attenuates collagen accumulation and dilation of the left ventricle (LV) post-myocardial infarction (MI); however the biomechanical mechanisms underlying the improved outcome are poorly understood. The aim of this study was to determine the mechanisms whereby MMP-9 deletion alters collagen network composition and assembly in the LV post-MI to modulate the mechanical properties of myocardial scar tissue. Adult C57BL/6J wild-type (WT; n=88) and MMP-9 null (MMP-9(-/-); n=92) mice of both sexes underwent permanent coronary artery ligation and were compared to day 0 controls (n=42). At day 7 post-MI, WT LVs displayed a 3-fold increase in end-diastolic volume, while MMP-9(-/-) showed only a 2-fold increase (p<0.05). Biaxial mechanical testing revealed that MMP-9(-/-) infarcts were stiffer than WT infarcts, as indicated by a 1.3-fold reduction in predicted in vivo circumferential stretch (p<0.05). Paradoxically, MMP-9(-/-) infarcts had a 1.8-fold reduction in collagen deposition (p<0.05). This apparent contradiction was explained by a 3.1-fold increase in lysyl oxidase (p<0.05) in MMP-9(-/-) infarcts, indicating that MMP-9 deletion increased collagen cross-linking activity. Furthermore, MMP-9 deletion led to a 3.0-fold increase in bone morphogenetic protein-1, the metalloproteinase that cleaves pro-collagen and pro-lysyl oxidase (p<0.05) and reduced fibronectin fragmentation by 49% (p<0.05) to enhance lysyl oxidase activity. We conclude that MMP-9 deletion increases infarct stiffness and prevents LV dilation by reducing collagen degradation and facilitating collagen assembly and cross-linking through preservation of the fibronectin network and activation of lysyl oxidase.

Expression of aromatase, androgen and estrogen receptors in peripheral target tissues in diabetes.

Our previous studies have shown that diabetes in the male streptozotocin (STZ)-induced diabetic rat is characterized by a decrease in circulating testosterone and concomitant increase in estradiol levels. Interestingly, this increase in estradiol levels persists even after castration, suggesting extra-testicular origins of estradiol in diabetes. The aim of the present study was to examine whether other target organs of diabetes may be sources of estradiol. The study was performed in male Sprague-Dawley non-diabetic (ND), STZ-induced diabetic (D) and STZ-induced diabetic castrated (Dcas) rats (n=8-9/group). 14 weeks of diabetes was associated with decreased testicular (ND, 26.3+/-4.19; D, 18.4+/-1.54; P<0.05), but increased renal (ND, 1.83+/-0.92; D, 7.85+/-1.38; P<0.05) and ocular (D, 23.4+/-3.66; D, 87.1+/-28.1; P<0.05) aromatase activity. This increase in renal (Dcas, 6.30+/-1.25) and ocular (Dcas, 62.7+/-11.9) aromatase activity persisted after castration. The diabetic kidney also had increased levels of tissue estrogen (ND, 0.31+/-0.01; D, 0.51+/-0.11; Dcas, 0.45+/-0.08) as well as estrogen receptor alpha protein expression (ND, 0.63+/-0.09; D, 1.62+/-0.28; Dcas, 1.38+/-0.20). These data suggest that in male STZ-induced diabetic rats, tissues other than the testis may become sources of estradiol. In particular, the diabetic kidney appears to produce estradiol following castration, a state that is associated with a high degree or renal injury. Overall, our data provides evidence for the extra-testicular source of estradiol that in males, through an intracrine mechanism, may contribute to the development and/or progression of end-organ damage associated with diabetes.

Early matrix metalloproteinase-9 inhibition post-myocardial infarction worsens cardiac dysfunction by delaying inflammation resolution

Matrix metalloproteinase-9 (MMP-9) is robustly elevated in the first week post-myocardial infarction (MI). Targeted deletion of the MMP-9 gene attenuates cardiac remodeling post-MI by reducing macrophage infiltration and collagen accumulation through increased apoptosis and reduced inflammation. In this study, we used a translational experimental design to determine whether selective MMP-9 inhibition early post-MI would be an effective therapeutic strategy in mice. We enrolled male C57BL/6J mice (3-6months old, n=116) for this study. Mice were subjected to coronary artery ligation. Saline or MMP-9 inhibitor (MMP-9i; 0.03μg/day) treatment was initiated at 3h post-MI and the mice were sacrificed at day (D) 1 or 7 post-MI. MMP-9i reduced MMP-9 activity by 31±1% at D1 post-MI (p<0.05 vs saline) and did not affect survival or infarct area. Surprisingly, MMP-9i treatment increased infarct wall thinning and worsened cardiac function at D7 post-MI. While MMP-9i enhanced neutrophil infiltration at D1 and macrophage infiltration at D7 post-MI, CD36 levels were lower in MMP-9i compared to saline, signifying reduced phagocytic potential per macrophage. Escalation and prolongation of the inflammatory response at D7 post-MI in the MMP-9i group was evident by increased expression of 18 pro-inflammatory cytokines (all p<0.05). MMP-9i reduced cleaved caspase 3 levels at D7 post-MI, consistent with reduced apoptosis and defective inflammation resolution. Because MMP-9i effects on inflammatory cells were significantly different from previously observed MMP-9 null mechanisms, we evaluated pre-MI (baseline) systemic differences between C57BL/6J and MMP-9 null plasma. By mass spectrometry, 34 plasma proteins were significantly different between groups, revealing a previously unappreciated altered baseline environment pre-MI when MMP-9 was deleted. In conclusion, early MMP-9 inhibition delayed inflammation resolution and exacerbated cardiac dysfunction, highlighting the importance of using translational approaches in mice.