Fatema Tuj Zohora, Shivani Arora, Alyssa Swiss, Naren Vyavahare
{"title":"使用靶向乙二胺四乙酸负载白蛋白纳米粒子逆转慢性肾病大鼠模型中的重度动脉钙化。","authors":"Fatema Tuj Zohora, Shivani Arora, Alyssa Swiss, Naren Vyavahare","doi":"10.21037/cdt-24-17","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Elastin degradation and severe calcification in the medial layer of the vessel wall, known as medial arterial calcification (MAC), is typical in the aging population and patients with metabolic disorders, such as diabetes and chronic kidney disease (CKD). We have previously reported that ethylene diamine tetraacetic acid (EDTA) delivery to the site of calcification can be achieved by tagging nanoparticles with an elastin antibody that recognizes explicitly damaged elastin, and such systemic therapy can remove focal calcium deposits from the calcified arteries in CKD rodent model. The current study aims to test whether heavy calcification seen throughout arterial tree and kidneys in CKD can be reversed with nanoparticle therapy.</p><p><strong>Methods: </strong>Thirty healthy male Sprague-Dawley rats weighing approximately 300 g, were placed on an adenine diet for 21 non-consecutive days to induce kidney failure, followed by daily vitamin D3 (VitD3) injections for 4 sequential days to cause severe calcification throughout the cardiovascular system and kidneys. DiR-dye loaded and elastin antibody conjugated albumin nanoparticles were used to confirm the targeting of nanoparticles to the calcification area. The rats were divided into two groups for targeted removal of calcification starting at day 7 of the last doses of VitD3. The experimental group received biweekly IV injections of anti-elastin antibody conjugated EDTA loaded human serum albumin nanoparticles (EDTA-HSA-El-Ab NPs), while the sham controls received blank nanoparticles (Blank-HSA-El-Ab NPs) (5 injections in total). Micro-computed tomography (microCT) was used to analyze the extent of calcification. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry studies were performed for osteogenic markers, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), and tissue non-specific alkaline phosphatase (TNAP). For comparison, aortic ring organ cultures from healthy rats were treated with high phosphate to induce calcification <i>in vitro</i>, and then they were treated with EDTA. Human calcified femoral arteries were also treated <i>ex vivo</i> with EDTA-HSA-EL-Ab NPs to test if nanoparticles remove heavy calcification.</p><p><strong>Results: </strong>EDTA-loaded nanoparticles that specifically target degraded elastin reversed existing heavy mineral deposits in arteries, as per elemental calcium analysis (124.161±34.410 µg Ca per mg of the dry aorta in Blank-HSA-El-Ab NPs <i>vs.</i> 100.520±19.131 µg in EDTA-HSA-El-Ab NPs group, P=0.04) and microCT (object volume, 129.001±37.785 <i>vs.</i> 29.815±24.169 mm<sup>3</sup>, P=0.0005). The reversal of aortic calcification was accompanied by a significant reduction of bone-associated mRNA expression of <i>BMP2</i> and <i>RUNX2</i> (P=0.01). Immunohistochemistry studies corroborated RT-PCR results, showing a reduction of BMP2 and RUNX2 stains in the vessel wall. The rat aortic ring culture study also showed similar results, where osteogenic genes (<i>BMP2, RUNX2</i>) and proteins (BMP2, RUNX2, TNAP) were suppressed upon reversal of calcification with EDTA (P=0.001). We also show <i>ex vivo</i> reversal of human femoral artery calcification by microCT (calcium intensity: untreated, 57.721±28.551 <i>vs.</i> day 6 of treatment, 5.441±3.615, P=0.01) by EDTA nanoparticle therapy.</p><p><strong>Conclusions: </strong>This is the first study showing the removal of calcium from heavily calcified arteries by using intravenous targeted EDTA therapy. Such therapy also reversed vascular smooth muscle cell osteoblastic transition and apoptosis in the arterial tissue, thereby potentially creating an environment for suitable tissue repair.</p>","PeriodicalId":9592,"journal":{"name":"Cardiovascular diagnosis and therapy","volume":null,"pages":null},"PeriodicalIF":2.1000,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11384463/pdf/","citationCount":"0","resultStr":"{\"title\":\"Reversal of heavy arterial calcification in a rat model of chronic kidney disease using targeted ethylene diamine tetraacetic acid-loaded albumin nanoparticles.\",\"authors\":\"Fatema Tuj Zohora, Shivani Arora, Alyssa Swiss, Naren Vyavahare\",\"doi\":\"10.21037/cdt-24-17\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Elastin degradation and severe calcification in the medial layer of the vessel wall, known as medial arterial calcification (MAC), is typical in the aging population and patients with metabolic disorders, such as diabetes and chronic kidney disease (CKD). We have previously reported that ethylene diamine tetraacetic acid (EDTA) delivery to the site of calcification can be achieved by tagging nanoparticles with an elastin antibody that recognizes explicitly damaged elastin, and such systemic therapy can remove focal calcium deposits from the calcified arteries in CKD rodent model. The current study aims to test whether heavy calcification seen throughout arterial tree and kidneys in CKD can be reversed with nanoparticle therapy.</p><p><strong>Methods: </strong>Thirty healthy male Sprague-Dawley rats weighing approximately 300 g, were placed on an adenine diet for 21 non-consecutive days to induce kidney failure, followed by daily vitamin D3 (VitD3) injections for 4 sequential days to cause severe calcification throughout the cardiovascular system and kidneys. DiR-dye loaded and elastin antibody conjugated albumin nanoparticles were used to confirm the targeting of nanoparticles to the calcification area. The rats were divided into two groups for targeted removal of calcification starting at day 7 of the last doses of VitD3. The experimental group received biweekly IV injections of anti-elastin antibody conjugated EDTA loaded human serum albumin nanoparticles (EDTA-HSA-El-Ab NPs), while the sham controls received blank nanoparticles (Blank-HSA-El-Ab NPs) (5 injections in total). Micro-computed tomography (microCT) was used to analyze the extent of calcification. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry studies were performed for osteogenic markers, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), and tissue non-specific alkaline phosphatase (TNAP). For comparison, aortic ring organ cultures from healthy rats were treated with high phosphate to induce calcification <i>in vitro</i>, and then they were treated with EDTA. Human calcified femoral arteries were also treated <i>ex vivo</i> with EDTA-HSA-EL-Ab NPs to test if nanoparticles remove heavy calcification.</p><p><strong>Results: </strong>EDTA-loaded nanoparticles that specifically target degraded elastin reversed existing heavy mineral deposits in arteries, as per elemental calcium analysis (124.161±34.410 µg Ca per mg of the dry aorta in Blank-HSA-El-Ab NPs <i>vs.</i> 100.520±19.131 µg in EDTA-HSA-El-Ab NPs group, P=0.04) and microCT (object volume, 129.001±37.785 <i>vs.</i> 29.815±24.169 mm<sup>3</sup>, P=0.0005). The reversal of aortic calcification was accompanied by a significant reduction of bone-associated mRNA expression of <i>BMP2</i> and <i>RUNX2</i> (P=0.01). Immunohistochemistry studies corroborated RT-PCR results, showing a reduction of BMP2 and RUNX2 stains in the vessel wall. The rat aortic ring culture study also showed similar results, where osteogenic genes (<i>BMP2, RUNX2</i>) and proteins (BMP2, RUNX2, TNAP) were suppressed upon reversal of calcification with EDTA (P=0.001). We also show <i>ex vivo</i> reversal of human femoral artery calcification by microCT (calcium intensity: untreated, 57.721±28.551 <i>vs.</i> day 6 of treatment, 5.441±3.615, P=0.01) by EDTA nanoparticle therapy.</p><p><strong>Conclusions: </strong>This is the first study showing the removal of calcium from heavily calcified arteries by using intravenous targeted EDTA therapy. Such therapy also reversed vascular smooth muscle cell osteoblastic transition and apoptosis in the arterial tissue, thereby potentially creating an environment for suitable tissue repair.</p>\",\"PeriodicalId\":9592,\"journal\":{\"name\":\"Cardiovascular diagnosis and therapy\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.1000,\"publicationDate\":\"2024-08-31\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11384463/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cardiovascular diagnosis and therapy\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.21037/cdt-24-17\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/8/23 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"CARDIAC & CARDIOVASCULAR SYSTEMS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cardiovascular diagnosis and therapy","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.21037/cdt-24-17","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/8/23 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"CARDIAC & CARDIOVASCULAR SYSTEMS","Score":null,"Total":0}
Reversal of heavy arterial calcification in a rat model of chronic kidney disease using targeted ethylene diamine tetraacetic acid-loaded albumin nanoparticles.
Background: Elastin degradation and severe calcification in the medial layer of the vessel wall, known as medial arterial calcification (MAC), is typical in the aging population and patients with metabolic disorders, such as diabetes and chronic kidney disease (CKD). We have previously reported that ethylene diamine tetraacetic acid (EDTA) delivery to the site of calcification can be achieved by tagging nanoparticles with an elastin antibody that recognizes explicitly damaged elastin, and such systemic therapy can remove focal calcium deposits from the calcified arteries in CKD rodent model. The current study aims to test whether heavy calcification seen throughout arterial tree and kidneys in CKD can be reversed with nanoparticle therapy.
Methods: Thirty healthy male Sprague-Dawley rats weighing approximately 300 g, were placed on an adenine diet for 21 non-consecutive days to induce kidney failure, followed by daily vitamin D3 (VitD3) injections for 4 sequential days to cause severe calcification throughout the cardiovascular system and kidneys. DiR-dye loaded and elastin antibody conjugated albumin nanoparticles were used to confirm the targeting of nanoparticles to the calcification area. The rats were divided into two groups for targeted removal of calcification starting at day 7 of the last doses of VitD3. The experimental group received biweekly IV injections of anti-elastin antibody conjugated EDTA loaded human serum albumin nanoparticles (EDTA-HSA-El-Ab NPs), while the sham controls received blank nanoparticles (Blank-HSA-El-Ab NPs) (5 injections in total). Micro-computed tomography (microCT) was used to analyze the extent of calcification. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry studies were performed for osteogenic markers, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), and tissue non-specific alkaline phosphatase (TNAP). For comparison, aortic ring organ cultures from healthy rats were treated with high phosphate to induce calcification in vitro, and then they were treated with EDTA. Human calcified femoral arteries were also treated ex vivo with EDTA-HSA-EL-Ab NPs to test if nanoparticles remove heavy calcification.
Results: EDTA-loaded nanoparticles that specifically target degraded elastin reversed existing heavy mineral deposits in arteries, as per elemental calcium analysis (124.161±34.410 µg Ca per mg of the dry aorta in Blank-HSA-El-Ab NPs vs. 100.520±19.131 µg in EDTA-HSA-El-Ab NPs group, P=0.04) and microCT (object volume, 129.001±37.785 vs. 29.815±24.169 mm3, P=0.0005). The reversal of aortic calcification was accompanied by a significant reduction of bone-associated mRNA expression of BMP2 and RUNX2 (P=0.01). Immunohistochemistry studies corroborated RT-PCR results, showing a reduction of BMP2 and RUNX2 stains in the vessel wall. The rat aortic ring culture study also showed similar results, where osteogenic genes (BMP2, RUNX2) and proteins (BMP2, RUNX2, TNAP) were suppressed upon reversal of calcification with EDTA (P=0.001). We also show ex vivo reversal of human femoral artery calcification by microCT (calcium intensity: untreated, 57.721±28.551 vs. day 6 of treatment, 5.441±3.615, P=0.01) by EDTA nanoparticle therapy.
Conclusions: This is the first study showing the removal of calcium from heavily calcified arteries by using intravenous targeted EDTA therapy. Such therapy also reversed vascular smooth muscle cell osteoblastic transition and apoptosis in the arterial tissue, thereby potentially creating an environment for suitable tissue repair.
期刊介绍:
The journal ''Cardiovascular Diagnosis and Therapy'' (Print ISSN: 2223-3652; Online ISSN: 2223-3660) accepts basic and clinical science submissions related to Cardiovascular Medicine and Surgery. The mission of the journal is the rapid exchange of scientific information between clinicians and scientists worldwide. To reach this goal, the journal will focus on novel media, using a web-based, digital format in addition to traditional print-version. This includes on-line submission, review, publication, and distribution. The digital format will also allow submission of extensive supporting visual material, both images and video. The website www.thecdt.org will serve as the central hub and also allow posting of comments and on-line discussion. The web-site of the journal will be linked to a number of international web-sites (e.g. www.dxy.cn), which will significantly expand the distribution of its contents.