Caterina Ivaldo’s research while affiliated with University of Genoa and other places

Background and Aims PCSK9 inhibitors (PCSK9i) are a new class of cholesterol-lowering drugs that is gaining market share in treating hypercholesterolemia because of their effectiveness in lowering LDL cholesterol and cardiovascular risk, even in patients with kidney disease. In addition to the well-known effect of inhibiting the LDL receptor catabolism, new pharmacological properties are being discovered. Experimental cellular and animal models have showed that PCSK9i reduce apoptosis, pyroptosis and in acute myocardial infarction decrease the infarct area, suggesting their protective role in tissue ischemia. However, so far PCSK9i effect on renal ischemic damage has not yet been studied. The aim of this study was to evaluate the effects of PEP 2-8 (PCSK9i) addition during hypothermic perfusion (HP) on ischemic kidney damage (ID) in a rat model of donation after circulatory death (DCD). Method n. 15 Sprague-Dawley rats were used as DCD donors. Briefly after a midline laparotomy, the left and right retroperitoneal renal areas were exposed, and lumbar arteries were isolated and sectioned; subsequently, the renal arteries and veins were isolated. After 30 min of warm ischemia, induced by aorta clamping, left and right nephrectomies were completed with preservation of the renal hilums. Then the kidneys were perfused with Perf-Gen Solution (100 ml) (Institut Georges Lopez, Lissieu, France) or Perf-Gen solution (100 ml) supplemented with 3ug (10ug/kg) of PEP 2-8. Continuous HP was performed for 6 h at 4 °C. DCD right and left kidneys of each rat were randomized to the following experimental groups: CTRL group: n = 15 DCD rat kidneys perfused with Perf-Gen PEP group: n = 15 DCD rat kidneys perfused with Pef-Gen supplemented with PEP 2-8. Effluent fluid was collected at the beginning (T0) and at the end (T6h) of HP. Glucose and potassium levels were measured in the effluent fluid at T0 and T6h. At T6h the kidneys were splitted into three aliquots; one was fixed in 10% formalin for morphological studies, and the other two were stored in trizol for gene expression studies and at −80 °C for biochemical assays respectively. Tubular ischemic damage (TID) was evaluated at T6 by scoring all tubules observed in at least 10 non-consecutive high-powered fields, as described by Paller (Paller Score). NOX- 4 gene expression was studied by RT-PCR assay. Tissue LDH and ATP levels were measured at T6h. Results PEP 2-8 treated kidneys showed a lower TID score compared to CTRL (PEP 107.0 IQR 90.75-116.0 vs CTRL 134.5 IQR 131.3-155.3 p < 0.005) (Fig. 1). The percentage of tubules with score zero (normal tubules) was higher in PEP than CTRL (PEP 29.0% range % 24.2-39.5 vs CTRL 9.5% range % 5.5-12.0, p < 0.001). The percentage of tubules with necrosis was lower in PEP than CTRL (PEP 5.5% range % 3.2-10.5 vs CTRL 12.5% range % 9.0-15.7, p < 0.005). Expression of NOX-4 gene, known inducer of apoptosis, was lower in PEP compared to CTRL (p < 0.05) Tissue ATP content was higher in PEP than CTRL (PEP 0.031 ± 0.01 vs CTRL 0.025 ± 0.006 p < 0.05). In tissue samples at T6 LDH was lower in PEP than CTRL (PEP 554.8 ± 164.7 vs CTRL 742.4 ± 256.7 p < 0.05) conversely the potassium release in effluent was significant only in CTRL (T6 vs T0 p < 0.01), according to TID score results. During HP, PEP 2-8 treated kidneys released more glucose in the effluent than CTRL kidneys (PEP 35.4 IQR 12.3-82.9 vs CTRL 12.3 IQR 7.1-70.5 p < 0.01). Conclusion Our preliminary results show that PEP 2-8 added to Perf-Gen solution during HP protects kidneys from ID, preserving energy metabolism in rat DCD model. To the best of our knowledge, this is the first evidence of protective role of PCSK9 inhibition in pre-transplant renal conditioning and potentially it could be used in conditioning other organs (liver, lung). However, further studies are necessary to understand the mechanisms underlying PCSK9i effect on energy metabolism and neoglucogenesis.

Background and Aims PCSK9 inhibitors (PCSK9i) are a new class of cholesterol-lowering drugs that is gaining market share in treating hypercholesterolemia because of their effectiveness in lowering LDL cholesterol and cardiovascular risk, even in patients with kidney disease. In addition to the well-known effect of inhibiting the LDL receptor catabolism, new pharmacological properties are being discovered. Experimental cellular and animal models have showed that PCSK9i reduce apoptosis, pyroptosis and in acute myocardial infarction decrease the infarct area, suggesting their protective role in tissue ischemia. However, so far PCSK9i effect on renal ischemic damage has not yet been studied. The aim of this study was to evaluate the effects of PEP 2-8(PCSK9i) addition during hypothermic perfusion (HP) on ischemic kidney damage (ID) in a rat model of donation after circulatory death (DCD). Method n. 15 Sprague-Dawley rats were used as DCD donors. Briefly after a midline laparotomy, the left and right retroperitoneal renal areas were exposed, and lumbar arteries were isolated and sectioned; subsequently, the renal arteries and veins were isolated. After 30 min of warm ischemia, induced by aorta clamping, left and right nephrectomies were completed with preservation of the renal hilums. Then the kidneys were perfused with Perf-Gen Solution (100 ml) (Institut Georges Lopez, Lissieu, France) or Perf-Gen solution (100 ml) supplemented with 3ug (10ug/kg) of PEP 2-8. Continuous HP was performed for 6 h at 4 °C. DCD right and left kidneys of each rat were randomized to the following experimental groups: CTRL group: n = 15 DCD rat kidneys perfused with Perf-Gen PEP group: n = 15 DCD rat kidneys perfused with Pef-Gen supplemented with PEP 2-8. Effluent fluid was collected at the beginning (T0) and at the end (T6h) of HP. Glucose and potassium levels were measured in the effluent fluid at T0 and T6h. At T6h the kidneys were splitted into three aliquots; one was fixed in 10% formalin for morphological studies, and the other two were stored in trizol for gene expression studies and at −80 °C for biochemical assays respectively. Tubular ischemic damage (TID) was evaluated at T6 by scoring all tubules observed in at least 10 non-consecutive high-powered fields, as described by Paller (Paller Score). NOX- 4 gene expression was studied by RT-PCR assay. Tissue LDH and ATP levels were measured at T6h. Results PEP 2-8 treated kidneys showed a lower TID score compared to CTRL (PEP 107.0 IQR 90.75-116.0 vs CTRL 134.5 IQR 131.3-155.3 p < 0.005) (Fig. 1). The percentage of tubules with score zero (normal tubules) was higher in PEP than CTRL (PEP 29.0% range % 24.2-39.5 vs CTRL 9.5% range % 5.5-12.0, p < 0.001). The percentage of tubules with necrosis was lower in PEP than CTRL (PEP 5.5% range % 3.2-10.5 vs CTRL 12.5% range % 9.0-15.7, p < 0.005). Expression of NOX-4 gene, known inducer of apoptosis, was lower in PEP compared to CTRL (p < 0.05) Tissue ATP content was higher in PEP than CTRL (PEP 0.031 ± 0.01 vs CTRL 0.025 ± 0.006 p < 0.05). In tissue samples at T6 LDH was lower in PEP than CTRL (PEP 554.8 ± 164.7 vs CTRL 742.4 ± 256.7 p < 0.05) conversely the potassium release in effluent was significant only in CTRL (T6 vs T0 p < 0.01), according to TID score results. During HP, PEP 2-8 treated kidneys released more glucose in the effluent than CTRL kidneys (PEP 35.4 IQR 12.3-82.9 vs CTRL 12.3 IQR 7.1-70.5 p < 0.01). Conclusion Our preliminary results show that PEP 2-8 added to Perf-Gen solution during HP protects kidneys from ID, preserving energy metabolism in rat DCD model. To the best of our knowledge, this is the first evidence of protective role of PCSK9 inhibition in pre-transplant renal conditioning and potentially it could be used in conditioning other organs (liver, lung). However, further studies are necessary to understand the mechanisms underlying PCSK9i effect on energy metabolism and neoglucogenesis.

The transcription factor NRF2 plays a crucial role in the regulation of antioxidant cell responses to stressors. It controls a plethora of genes with pro-surviving activity, among which heme oxygenase 1 (HO-1) has been recognized to play a potent anti-inflammatory activity through its metabolites carbon monoxide (CO) and bilirubin. Macrophages trigger inflammation by recognizing pathogens and tissue damage using different toll like receptors (TLRs) that through the activation of complex signalling pathways lead to specific cell responses. In particular, TLR4 and TLR7/8 have been proved to have downstream signal cascades highly interrelated, involving both NF-kB and interferon regulatory factor 5 (IRF5) transcription factors and leading to pro-inflammatory cytokine release (e.g. TNF-α). Interestingly, IRF5 has been proved to be dysregulated in autoimmune diseases, cancer, cardiovascular diseases and neuroinflammation. Thus, our work aims at understanding the involvement of NRF2/HO-1 in the regulation of IRF5, which has never been investigated.

Classical cadherins, including vascular endothelial (VE)-cadherin, are targeted by matrix metalloproteinases (MMPs) and γ-secretase during adherens junction (AJ) disassembly, a mechanism that might have relevance for endothelial cell (EC) integrity and vascular homeostasis. Here, we show that oxidative stress triggered by H2O2 exposure induced efficient VE-cadherin proteolysis by MMPs and γ-secretase in human umbilical endothelial cells (HUVECs). The cytoplasmic domain of VE-cadherin produced by γ-secretase, VE-Cad/CTF2—a fragment that has eluded identification so far—could readily be detected after H2O2 treatment. VE-Cad/CTF2, released into the cytosol, was tightly regulated by proteasomal degradation and was sequentially produced from an ADAM10/17-generated C-terminal fragment, VE-Cad/CTF1. Interestingly, BMP9 and BMP10, two circulating ligands critically involved in vascular maintenance, significantly reduced VE-Cad/CTF2 levels during H2O2 challenge, as well as mitigated H2O2-mediated actin cytoskeleton disassembly during VE-cadherin processing. Notably, BMP9/10 pretreatments efficiently reduced apoptosis induced by H2O2, favoring endothelial cell recovery. Thus, oxidative stress is a trigger of MMP- and γ-secretase-mediated endoproteolysis of VE-cadherin and AJ disassembly from the cytoskeleton in ECs, a mechanism that is negatively controlled by the EC quiescence factors, BMP9 and BMP10.

Classical cadherins, including vascular endothelial (VE)-cadherin, are targeted by matrix metalloproteinases (MMPs) and γ-secretase during adherens junction (AJ) disassembly, a mechanism that might have relevance for endothelial cell (EC) integrity and vascular homeostasis. Here, we show that oxidative stress triggered by H2O2 exposure induced efficient VE-cadherin proteolysis by MMPs and γ-secretase in human umbilical endothelial cells (HUVECs). The cytoplasmic domain of VE-cadherin produced by γ-secretase, VE-Cad/CTF2 - a fragment that has eluded identification so far - could readily be detected after H2O2 treatment. VE-Cad/CTF2, released into the cytosol, was tightly regulated by proteasomal degradation and was sequentially produced from an ADAM10/17-generated C-terminal fragment, VE-Cad/CTF1. Interestingly, BMP9 and BMP10, two circulating ligands critically involved in vascular maintenance, significantly reduced VE-Cad/CTF2 levels during H2O2 challenge, as well as mitigated H2O2- mediated actin cytoskeleton disassembly during VE-cadherin processing. Notably, BMP9/10 pretreatments efficiently reduced apoptosis induced by H2O2, favoring endothelial cell recovery. Thus, oxidative stress is a trigger of MMP- and γ-secretase-mediated endoproteolysis of VE-cadherin and AJ disassembly from the cytoskeleton in ECs, a mechanism that is negatively controlled by the EC quiescence factors, BMP9 and BMP10.

Induction of heme oxygenase 1 (HO-1) favors immune-escape in BRAFV600 melanoma cells treated with Vemurafenib/PLX4032 under standard cell culture conditions. However, the oxygen tension under standard culture conditions (~18 kPa O2) is significantly higher than the physiological oxygen levels encountered in vivo. In addition, cancer cells in vivo are often modified by hypoxia. In this study, MeOV-1 primary melanoma cells bearing the BRAFV600E mutation, were adapted to either 5 kPa O2 (physiological normoxia) or 1 kPa O2 (hypoxia) and then exposed to 10 μM PLX4032. PLX4032 abolished ERK phosphorylation, reduced Bach1 expression and increased HO-1 levels independent of pericellular O2 tension. Moreover, cell viability was significantly reduced further in cells exposed to PLX4032 plus Tin mesoporphyrin IX, a HO-1 inhibitor. Notably, our findings provide the first evidence that HO-1 inhibition in combination with PLX4032 under physiological oxygen tension and hypoxia restores and increases the expression of the NK ligands ULBP3 and B7H6 compared to cells exposed to PLX4032 alone. Interestingly, although silencing NRF2 prevented PLX4032 induction of HO-1, other NRF2 targeted genes were unaffected, highlighting a pivotal role of HO-1 in melanoma resistance and immune escape. The present findings may enhance translation and highlight the potential of the HO-1 inhibitors in the therapy of BRAFV600 melanomas.

Heme oxygenase 1 (HO-1) plays a key role in cell adaptation to stressors through the antioxidant, antiapoptotic, and anti-inflammatory properties of its metabolic products. For these reasons, in cancer cells, HO-1 can favor aggressiveness and resistance to therapies, leading to poor prognosis/outcome. Genetic polymorphisms of HO-1 promoter have been associated with an increased risk of cancer progression and a high degree of therapy failure. Moreover, evidence from cancer biopsies highlights the possible correlation between HO-1 expression, pathological features, and clinical outcome. Indeed, high levels of HO-1 in tumor specimens often correlate with reduced survival rates. Furthermore, HO-1 modulation has been proposed in order to improve the efficacy of antitumor therapies. However, contrasting evidence on the role of HO-1 in tumor biology has been reported. This review focuses on the role of HO-1 as a promising biomarker of cancer progression; understanding the correlation between HO-1 and clinical data might guide the therapeutic choice and improve the outcome of patients in terms of prognosis and life quality.