Picrosirius Red Staining Research Articles

Cardioprotective Role of Beta Hydroxybutyrate in Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD)

Background: Cardiovascular disease (CVD) is the leading cause of mortality in metabolic dysfunction-associated steatotic liver disease (MASLD). Beta hydroxybutyrate (BHOB), a liver metabolite, is the major ketone that serves as an alternative fuel source in the body. Previously, we observed cardiovascular dysfunction that was associated with reduced circulating BHOB in hepatocyte-specific PPARα knockout mice (PparaHEPKO), a mouse model that exhibits hepatic steatosis independent of obesity and insulin resistance. Hypothesis: We hypothesize that restoring plasma BHOB levels will attenuate the mechanisms underlying hepatic steatosis-induced cardiovascular dysfunction and improve cardiovascular function in PparaHEPKO mice.Aims: To determine the cardioprotective role of increased plasma levels of BHOB in CVDs induced by MASLD. Methods: 30 week old male PparaHEPKO mice were given 1,3 butanediol (20% in drinking water) (PparaHEPKO+ 1,3 butanediol) or vehicle (PparaHEPKO) (n=6) for 6 weeks. Plasma BHOB was measured at baseline and after treatment. Cardiac structure and function were measured by high resolution ultrasound echocardiography (VEVO 3100). Mean arterial blood pressure was measured by radio telemetry. Cardiac lipid accumulation was determined by Oil Red O (ORO) and cardiac triglyceride levels. Cardiac apoptosis and fibrosis were determined by TUNEL and picrosirius red staining, further confirmed by western blot. Cardiac natriuretic peptides were determined by real-time PCR. Liver fat was determined by EchoMRI, ORO staining and hepatic triglyceride levels.Results: After 6 weeks of 1,3 butanediol treatment, PparaHEPKO exhibit increased plasma BHOB compared to baseline (0.5 ± 0.01 vs. 0.2 ± 0.02mmol/L), attenuated arterial blood pressure compared to control (109 ± 3 vs. 121 ± 4mmHg), improved cardiac output (13.8 ± 0.8 vs. 11.1 ± 0.7mL/min), stroke volume (31.1 ± 2.1 vs. 23.4 ± 1.3μL), and isovolumic relaxation time (18.7 ± 0.8 vs. 20.6 ± 0.9ms). 1,3 butanediol treatment also attenuated vascular stiffness, cardiac lipid (0.7 ± 0.27 vs. 1.5 ± 0.17), ANP (1.1 ± 0.03 vs. 1.3 ± 0.03), COL1A1 (0.9 ± 0.1 vs. 9.0 ± 0.5), and cleaved caspase-3 (1.8 ± 0.3 vs. 3.7 ± 0.7). Interestingly, 1,3 butanediol did not alleviate hepatic fat compared to control as demonstrated by EchoMRI (0.8 ± 0.3 vs. 0.7 ± 0.3%), hepatic triglyceride (1.4 ± 0.3 vs. 1.3 ± 0.2mM) and Oil Red O staining.Conclusion: Our findings indicate that increasing plasma BHOB level improves arterial blood pressure, exercise tolerance, systolic, diastolic, and vascular functions in MASLD-induced CVD. Furthermore, BHOB attenuates cardiac lipid, apoptosis and fibrosis. However, BHOB did not alleviate hepatic steatosis suggesting that BHOB improves cardiovascular functions in PparaHEPKO mice independent of hepatic fat content. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant 1R01DK121748-01A1 (D.E. Stec), the National Institute of General Medical Sciences Grant P20GM104357-02, P30GM149404 and P20GM144041 (D.E. Stec), and by American Heart Association Postdoctoral Award (23POST1020493, O.O. Badmus). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Macrophage to Myofibroblast Transition Promotes Hypertension-Induced Cardiac Diastolic Dysfunction

Cardiac diastolic dysfunction is a pathological continuum of hypertension eventually leading to heart failure—which is responsible for ~13% of all deaths in the United States. A hallmark of this disease is cardiac fibrotic remodeling, in which excessive extracellular matrix components are deposited into the myocardium. Recent studies have shown the novel ability of macrophages to differentiate into a fibrotic myofibroblast-like phenotype—termed macrophage to myofibroblast transition (MMT). This phenomenon has been implicated in renal fibrosis but, has not been investigated in the heart during hypertension or diastolic dysfunction. Therefore, we hypothesize that MMT plays a causal role in cardiac fibrotic remodeling in hypertension, promoting diastolic dysfunction. As such, we seek to investigate the prevalence of myofibroblast-transitioned macrophages in the heart, as well as determine the gravity of MMT-mediated fibrotic cardiac remodeling. To understand the pathological progression of hypertension to diastolic dysfunction, we utilized the classic DOCA + NaCl model of hypertension in C57BL/6 mice. This investigation is multi-pronged, in that, we seek to gauge diastolic function in vivo, as well as investigate the microenvironment of the heart in primary in vitro experiments. To achieve this, we employ echocardiography and direct left ventricular pressure and volume measurements to determine the progression of hypertension to diastolic dysfunction in the adult mouse heart. For primary in vitro analyses, mouse hearts were isolated and digested, and leukocytes were isolated by Percoll gradient. Cells were stained with α-SMA (myofibroblast) and CD68 (monocyte/macrophage). Finally, the deposition of fibrotic collagen was assessed by both Masson’s trichrome and picrosirius red tissue stains. Mice were subjected to DOCA + salt to induce hypertension. Ten days after this treatment, the left ventricular pressure change as a function of time was measured in the left ventricle. This analysis revealed degraded heart function, realized by a significant decrease in -dp/dt as soon as ten days following the onset of hypertension. Following, another cohort of hypertensive WT mouse hearts was isolated for staining. The results of these assays revealed increased cardiac fibrosis in the hearts of hypertensive mice—likely contributing to the deficit in cardiac function. Finally, in addition to this, flow cytometric analyses of isolated cardiac leukocytes revealed that hypertensive mice had a significant increase in the α-SMA+/CD68+ population of cells in the hypertensive mouse heart, implicating MMT in the rapid pathological progression of hypertension to diastolic dysfunction. These results characterize the presence of MMT cells in the hypertensive mouse heart. Furthermore, the increase of myofibroblast transition is accompanied by decreased cardiac diastolic function and enhanced fibrotic remodeling in the heart—alluding to the role of MMT in the pathological progression of hypertension to diastolic dysfunction. Future efforts into this phenomenon involve the real-time progression of hypertension to diastolic dysfunction via echocardiography, as well as a final, definitive measurement of diastolic function by PV loop. Finally, we will employ an angiotensin II model of hypertension to ensure that this phenomenon is encapsulated in other forms of hypertension, rather than just the DOCA + Salt model. We thank our sources of funding:NIG R01-HL146713, AHA 23TPA1076467. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of CD8 T-cell transfer on biomechanics of post-myocardial infarction scar formation

CD8+ T-cells are adverse regulators of post-MI remodeling, resulting in attenuated cardiac function and overall survival. Based on previous studies, we hypothesize that CD8+ T-cells impair cardiac function by altering scar composition. MI was induced by ligating the left anterior descending coronary artery on C57BL6/J wildtype (WT; 3-7 months of age, n≥2/sex) and CD8atm1mak (CD8−/−; 3-7 months of age, n≥2/sex/ group) mice. CD8−/− mice were injected with either vehicle or naïve splenic CD8+ T-cells via tail vein, 4-hours post-coronary artery ligation to assess potential effects on the scar. On day 7 post-MI, infarct tissue was collected, undergoing either passive stretch biomechanical analysis or histological and biochemical assays for collagen composition. Effects of granzyme (Gzm) B and K on collagen cleavage were tested using a fluorogenic collagen cleavage assay to examine possible mechanisms of scar alteration. Mice lacking CD8+ T-cells had improved ejection fraction and decreased dilation compared to WT mice at post-MI Day 7. This protective effect was lost in CD8−/− mice that received splenic CD8+ T-cells. Biomechanical analysis demonstrated CD8−/− mice had a 2-fold increase in regional stiffness compared to WT mice. Re-supplementation with splenic CD8+ T-cells decreased scar tissue stiffness mimicking biomechanics of WT mice. Picrosirius red staining surprisingly showed no significant differences in total collagen levels (p=0.51). Immunoblotting for Collagen 1 trended an increase in the 250 kDa band in CD8−/− mice that decreased after supplementation indicating a loss of pro-collagen formation (p=0.051). Ex-vivo cleavage assay demonstrated GzmK cleaved collagen in a concentration (0-50 AU) and temporal-dependent manner (0-24 hrs). Moreover, GzmK demonstrated a 10-fold greater capacity in collagen cleavage compared to GzmB at 25 AU, indicating the role GzmK plays during post-MI remodeling may be distinctly separate from that of GzmB. In conclusion, our data demonstrates that CD8+ T-cells regulate cardiac fibrosis potentially in part through the release of granzymes, leading to alterations in biomechanical capability of the left ventricle. This work was supported by the National Institutes of Health T32GM123055, R25GM113278; the Biomedical Laboratory Research and Development Service of the Veterans Affairs Offce of Research and Development Award IK2BX003922 and BX003922; and South Carolina Translational Research Center UL1TR001450. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3038: Platelet-specific Deletion Of Transforming Growth Factor-β1 Augments Angiotensin Ii-induced Abdominal Aortic Aneurysm In Mice

Background: Transforming growth factor beta (TGFβ) blockade via systemic antibody targeting has been shown to augment murine AAA progression in multiple independent studies. Despite these findings, the primary source of protective TGFβ within AAA remains unclear. Previous research conducted in our laboratory demonstrates deletion of platelet-specific TGFβ1 exacerbates aneurysms in the elastase mouse model of AAA. Our present study extends these results by showing a comparable phenotype in the angiotensin II (Ang II) murine model of AAA. Methods and Results: Low density lipoprotein receptor deficient ( Ldlr -/- ) mice with platelet-specific deletion of Tgfβ1 were created by breeding male mice expressing Cre recombinase under the control of a Pf4 promoter to female Tgfβ1 floxed mice (Jackson Labs). Male mice were fed high fat diet 1 week prior to and throughout subcutaneous infusion of AngII (1,000 ng/kg/min). After day 28 of infusion, ex vivo aortic diameter was significantly increased in Cre+ (n = 17) vs Cre- (n = 18) mice ( Cre- : 1.75 ± 0.17 mm; Cre+ : 2.43 ± 0.21 mm; P < 0.05). AAA incidence, defined as aortic diameter ≥ 1.2mm, was 56% for Cre- mice vs. 93% in the Cre+ group. Histological analysis of picrosirius red staining revealed a marked reduction in type 1 abdominal collagen deposition as a proportion of total cellular area in aortas from Cre+ mice versus Cre- controls. OCT embedded sections of aneurysms from both cohorts were also probed for macrophage marker CD68 and analyzed using immunofluorescent microscopy. We found Cre+ aortas had significantly increased macrophage infiltration compared to Cre- aortas. Conclusions: This study of platelet-specific deletion of TGFβ1 in a secondary model of murine AAA supports the conclusion that TGFβ1 sourced form platelets plays a critically important role in the protection of AAA.

PTSD-like mice display negative cardiac characteristics post-inescapable foot shock

Post-traumatic stress disorder (PTSD) is a risk factor for cardiovascular disease, however the mechanism behind this correlation is unknown. We hypothesized that PTSD alters cardiac homeostasis by increasing macrophage numbers and initiating cardiac fibrosis. To induce experimental PTSD, male and female C57BL/6 mice (4-weeks: n=16-17/sex, males=4.7±0.4 months old; females=4.8±0.3 months old; 8-weeks: n=13-16/sex, males=6.0±0.3 months old; females=6.3±0.5 months old) were exposed to 5 separate foot-shock incidences (IFS; 1.0 mA, 1 sec duration) in 6 min. Control mice (n=8-9/sex) were placed in the same chambers but experienced no foot shocks. Behavioral tests (16 parameters based on the DSM-5 clinical standards) were performed to characterize mice that do not demonstrate PTSD-like behavioral characteristics (NR) and PTSD-like mice. Doppler echocardiography measurements were collected serially up to 8-weeks post-IFS. Histological and gene assessments were collected 4- and 8-weeks post-IFS to measure markers of adverse cardiac remodeling that resulted in later functional changes. Over time PTSD-like mice demonstrated a decline in cardiac function becoming significantly impaired by 8-weeks as shown by an increase in E/e’ ratio along with left atrial diameter compared to controls, indicative of impaired LV filling pressure and diastolic dysfunction (p<0.05 for all). Beginning at 4-weeks post-IFS, PTSD-like mice had increased picrosirius red staining (PSR) compared to control mice (p<0.05) that remained elevated at 8-weeks. PTSD-like mice had increased macrophage numbers (Mac3) at 4-weeks post-IFS compared to control mice (p<0.05) and showed an increased trend when compared to controls 8-weeks post-IFS (p=0.09). To determine potential signaling mechanisms, Ccl2, Ccl5, and discoidin domain receptor tyrosine kinase 2 (DDR2) mRNA levels were assessed. Gene expression of Ccl2 demonstrated a decreased trend in PTSD-like mice (p=0.13), while Ccl5 (p=0.08) and DDR2 (p=0.06) mRNA demonstrated an increased trend in PTSD-like mice when compared to controls. Our data indicates that in a mouse model of PTSD, animals that demonstrate PTSD-like behaviors show signs of increased cardiac inflammation stimulating cardiac remodeling and cardiac dysfunction. This work was supported by the National Institutes of Health T32GM123055; the American Heart Association Innovator Project IPA35260039, GM113278; the Biomedical Laboratory Research and Development Service of the Veterans Affairs Offce of Research and Development Award IK2BX003922, I01BX00584; and South Carolina Translational Research Center UL1TR001450. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adenomatoid odontogenic tumor: A histopathologic profile of 43 cases with evidence supporting a mixed odontogenic origin

BackgroundAdenomatoid Odontogenic Tumor (AOT) accounts for 3% of all odontogenic tumors. It has been classified by WHO as an odontogenic tumor of purely epithelial origin. The current study attempts to establish the origin of the tumor along with detailed histopathological and clinicoradiographic analysis of 43 cases of AOT. Material and methodsForty-three cases were reviewed from the departmental archives for demographic data, radiographic features and histological features. Further, histopathological slides were stained with Picrosirius Red (PSR) and observed under polarised light. ResultsA majority of the cases were seen in the anterior jaws (76.7%), and were less than 3 cms (76.7%) in greatest dimension. Equal number of cases were of follicular and extra-follicular location while one was peripheral. Predominantly solid histological pattern was noted in 53.5%. Varied sub-patterns were observed with most cases exhibiting solid nodules and strands of tumor cells. Few cases showed melanin pigmentation. Over a third of cases (37.2%) showed dentigerous cyst like areas and one case each showed features of ossifying fibroma and focal cemento-osseous dysplasia. Tumor droplets, hyaline rings within duct-like structures, dentinoid material and osteodentin showed reddish yellow birefringence when observed under polarised microscopy post PSR staining. ConclusionThis study highlights the diverse histopathological variation of AOT with evidence to reclassify it as a mixed odontogenic tumor based on the polarising microscopic findings with PSR staining.

Long-Term Protective Effects of Succinate Dehydrogenase Inhibition during Reperfusion with Malonate on Post-Infarction Left Ventricular Scar and Remodeling in Mice.

Succinate dehydrogenase inhibition with malonate during initial reperfusion reduces myocardial infarct size in both isolated mouse hearts subjected to global ischemia and in in situ pig hearts subjected to transient coronary ligature. However, the long-term effects of acute malonate treatment are unknown. Here, we investigated whether the protective effects of succinate dehydrogenase inhibition extend to a reduction in scar size and adverse left ventricular remodeling 28 days after myocardial infarction. Initially, ten wild-type mice were subjected to 45 min of left anterior descending coronary artery (LAD) occlusion, followed by 24 h of reperfusion, and were infused during the first 15 min of reperfusion with saline with or without disodium malonate (10 mg/kg/min, 120 μL/kg/min). Malonate-treated mice depicted a significant reduction in infarct size (15.47 ± 3.40% of area at risk vs. 29.34 ± 4.44% in control animals, p < 0.05), assessed using triphenyltetrazolium chloride. Additional animals were then subjected to a 45 min LAD ligature, followed by 28 days of reperfusion. Treatment with a single dose of malonate during the first 15 min of reperfusion induced a significant reduction in scar area, measured using Picrosirius Red staining (11.94 ± 1.70% of left ventricular area (n = 5) vs. 23.25 ± 2.67% (n = 9), p < 0.05), an effect associated with improved ejection fraction 28 days after infarction, as determined using echocardiography, and an attenuated enhancement in expression of the pro-inflammatory and fibrotic markers NF-κB and Smad2/3 in remote myocardium. In conclusion, a reversible inhibition of succinate dehydrogenase with a single dose of malonate at the onset of reperfusion has long-term protective effects in mice subjected to transient coronary occlusion.

Factors Affecting the Evaluation of Collagen Deposition and Fibrosis In Vitro.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Excessive fat expenditure in MCT-induced heart failure rats is associated with BMAL1/REV-ERBα circadian rhythmic loop disruption

Fat loss predicts adverse outcomes in advanced heart failure (HF). Disrupted circadian clocks are a primary cause of lipid metabolic issues, but it's unclear if this disruption affects fat expenditure in HF. To address this issue, we investigated the effects of disruption of the BMAL1/REV-ERBα circadian rhythmic loop on adipose tissue metabolism in HF.50 Wistar rats were initially divided into control (n = 10) and model (n = 40) groups. The model rats were induced with HF via monocrotaline (MCT) injections, while the control group received equivalent solvent injections. After establishing the HF model, the model group was further subdivided into four groups: normal rhythm (LD), inverted rhythm (DL), lentivirus vector carrying Bmal1 short hairpin RNA (LV-Bmal1 shRNA), and empty lentivirus vector control (LV-Control shRNA) groups, each with 10 rats. The DL subgroup was exposed to a reversed light–dark cycle of 8 h: 16 h (dark: light), while the rest adhered to normal light–dark conditions (light: dark 12 h: 12 h). Histological analyses were conducted using H&E, Oil Red O, and Picrosirius red stains to examine adipose and liver tissues. Immunohistochemical staining, RT-qPCR, and Western blotting were performed to detect markers of lipolysis, lipogenesis, and beiging of white adipose tissue (WAT), while thermogenesis indicators were detected in brown adipose tissue (BAT). The LD group rats exhibited decreased levels of BMAL1 protein, increased levels of REV-ERBα protein, and disrupted circadian circuits in adipose tissue compared to controls. Additionally, HF rats showed reduced adipose mass and increased ectopic lipid deposition, along with smaller adipocytes containing lower lipid content and fibrotic adipose tissue. In the LD group WAT, expression of ATGL, HSL, PKA, and p-PKA proteins increased, alongside elevated mRNA levels of lipase genes (Hsl, Atgl, Peripilin) and FFA β-oxidation genes (Cpt1, acyl-CoA). Conversely, lipogenic gene expression (Scd1, Fas, Mgat, Dgat2) decreased, while beige adipocyte markers (Cd137, Tbx-1, Ucp-1, Zic-1) and UCP-1 protein expression increased. In BAT, HF rats exhibited elevated levels of PKA, p-PKA, and UCP-1 proteins, along with increased expression of thermogenic genes (Ucp-1, Pparγ, Pgc-1α) and lipid transportation genes (Cd36, Fatp-1, Cpt-1). Plasma NT-proBNP levels were higher in LD rats, accompanied by elevated NE and IL-6 levels in adipose tissue. Remarkably, morphologically, the adipocytes in the DL and LV-Bmal1 shRNA groups showed reduced size and lower lipid content, while lipid deposition in the liver was more pronounced in these groups compared to the LD group. At the gene/protein level, the BMAL1/REV-ERBα circadian loop exhibited severe disruption in LV-Bmal1 shRNA rats compared to LD rats. Additionally, there was increased expression of lipase genes, FFA β oxidation genes, and beige adipocyte markers in WAT, as well as higher expression of thermogenic genes and lipid transportation genes in BAT. Furthermore, plasma NT-proBNP levels and adipose tissue levels of NE and IL-6 were elevated in LV-Bmal1 shRNA rats compared with LD rats. The present study demonstrates that disruption of the BMAL1/REV-ERBα circadian rhythmic loop is associated with fat expenditure in HF. This result suggests that restoring circadian rhythms in adipose tissue may help counteract disorders of adipose metabolism and reduce fat loss in HF.

557 Dual TGFβR1/MAP4K4 inhibitor reduces kidney injury in a mouse model of renal fibrosis.

OBJECTIVES/GOALS: Renal fibrosis is a critical pathophysiological event in chronic kidney diseases. Our goal is to determine the ability of dual-inhibitor of transforming growth factor beta receptor 1 (TGFα²R1) and mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), TK850, on reducing kidney fibrosis. METHODS/STUDY POPULATION: To test the renal anti-fibrotic action ofdual TK850,8-10-week-old male and female C57BL/6mice with unilateral ureteral obstruction (UUO) induced kidney fibrosis were used. Mice were separated into 3 groups: group 1 contained mice that had UUO surgery (UUO control), group 2 contained mice prophylactically treated with TK850 thatstarted 7 days prior to UUO(UUO-P,20 mpk/d/ip), and group 3 contained mice interventionally treated with TK850 that started3 days after UUO(UUO-I,20 mpk/d/ip). Ten days following UUO the kidneys and blood were collected for analysis. Renal fibrosis was assessed from hydroxyproline content (measure of collagen)and histological collagen analysis using Picrosirius red stain. RESULTS/ANTICIPATED RESULTS: Renal hydroxyproline was increased equally in the UUO kidney of male (5.4 ± 0.41 µg/10mg, n=5) and female mice (5.5 ± 0.50 µg/10mg, n=5) compared to the contralateral control kidney (2.9 ± 0.14 µg/10mg, n=10). TK850 treatment in UUO-P mice (n=10, 3.4 ± 0.24 µg/10mg) and UUO-I mice (4.30 ± 0.20 µg/10mg, n=10) had significantly reduced hydroxyproline levels. Histopathological evaluation revealed that kidney injury increased collagen deposition in the UUO kidney (17.1 ± 0.43% collagen positive area, n=10) compared to the control kidney (2.0 ± 0.23%, n=10). TK850 treatment in UUO-P mice significantly attenuated collagen deposition (10.5 ± 0.38%, n=10), while UUO-I had significantly reduced collagen deposition as well (13.1 ± 0.25%, n=10). DISCUSSION/SIGNIFICANCE: Taken together, these results validate the dual TGFβR1/MAP4K4 inhibitor, TK850 as a potential therapeutic to mitigate renal fibrosis and supports the emergence of a combinational pharmacotherapeutic approach for multi-factorial kidney diseases.

Ablation of Brg1 in fibroblast/myofibroblast lineages attenuates renal fibrosis in mice with diabetic nephropathy

AimsDiabetic nephropathy (DN) is one of the most common complications of diabetes and represents a prototypical form of chronic kidney disease (CKD). Interstitial fibrosis is a key pathological feature of DN. During DN-associated renal fibrosis, resident fibroblasts trans-differentiate into myofibroblasts to remodel the extracellular matrix, the underlying epigenetic mechanism of which is not entirely clear. MethodsDiabetic nephropathy was induced in C57B6/j mice by a single injection with streptozotocin (STZ). Gene expression was examined by quantitative PCR and Western blotting. Renal fibrosis was evaluated by PicroSirius Red staining. ResultsWe report that expression of Brg1, a chromatin remodeling protein, in renal fibroblasts was up-regulated during DN pathogenesis as assessed by single-cell RNA-seq. Treatment with high glucose similarly augmented Brg1 expression in primary renal fibroblasts in vitro. Importantly, Brg1 ablation in quiescent renal fibroblasts or in mature myofibroblasts equivalently attenuated renal fibrosis in the context of diabetic nephropathy in mice. Additionally, administration with a small-molecule Brg1 inhibitor PFI-3 ameliorated renal fibrosis and improved renal function in mice induced to develop DN. SignificanceIn conclusion, our data provide novel genetic evidence that links Brg1 to fibroblast-myofibroblast transition and renewed rationale for targeting Brg1 in the intervention of DN-associated renal fibrosis.

Abstract 5571: Collagen stabilization directs immune suppression in the head and neck squamous cell carcinoma hypoxic subtype

Abstract Head &amp; neck squamous cell carcinoma (HNSCC) show limited clinical benefit to immune checkpoint inhibitors (ICI). Interactions between extracellular matrix (ECM), tumor, and immune cells underly the mechanisms leading to immunosuppression and resistance to ICIs. Collagen matrix remodeling within tumor stroma can influence accessibility and function of immune cells. Specifically, telopeptidyl lysine residues in fibrillar collagen can be hydroxylated by Lysyl hydroxylase 2 (LH2) prior to cross-linking which leads to stiffened, stable collagen. We previously showed that this LH2-modified tumor microenvironment (TME) drives HNSCC invasion and metastasis. Here, we elucidate the interplay between LH2-modified collagen and the immune landscape within TME of hypoxic and immunosuppressive HNSCC. Bioinformatics analyses of RNAseq datasets were performed to assess pathways regulating collagen deposition and stabilization. Tissue microarray having 81 primary, 28 recurrent primary, and 3 lymph node metastases tumor cores was analyzed. Cores were classified according to our published Hypoxia-Immune signature (Chaudhary R. et al. 2023, Can Res Comm). We performed Second Harmonic Generation imaging of H&amp;E stained TMA section to analyze the orientation and length of collagen fibrils. Picrosirius red (PSR) staining allowed quantification of total collagen and collagen maturation was assessed by polarized light imaging of birefringence. Further, we performed multiplex immunofluorescence staining for CD3+ (T-cell); CD14+ (Monocyte &amp; Macrophage); CD11c+ (Dendritic); Pax5+ (B-cell); Pan-Cytokeratin+ (tumor), and LH2. Low (0-1) LH2 expression was seen in 29.5% of analyzed cores whereas 70.5% of cores scored high (2-3) for LH2 expression. Cores classified under the hypoxia subtype exhibit high LH2 and enrichment of collagen stability signature. Late-stage tumors have highly oriented collagen fibers compared to normal tissue or early-stage tumors which have randomly distributed fibers. CT-Fire-based measurements revealed that LH2hi cores have significantly increased fiber length and lower coefficient of variance for fiber angles. The area of collagen measured by PSR staining had no correlation with tumor stage or subtype indicating that collagen deposition is similarly high in HNSCCs. However, birefringence measurements showed that a higher fraction of mature fibrillar collagen is associated with late-stage LH2hi tumors. Interestingly, LH2 expression positively correlated with increased CD3+ T cells in stroma suggesting that anti-tumor T cells are physically obstructed by the presence of LH2-stabilized and stiffened collagen fibers. LH2 expression influences the quality of collagen but not the quantity. Understanding the effect of LH2-mediated collagen modifications on the TIME may have implications for therapeutic strategies to enhance anti-tumor immune response in HNSCC. Citation Format: Sonal Srivastava, John Powers, Ritu Chaudhary, Sadegh Marzban, Robbert J. Slebos, Jeffrey West, Xiaofei Song, Jose A. Guevara-Patino, Christine H. Chung, Antonio L. Amelio. Collagen stabilization directs immune suppression in the head and neck squamous cell carcinoma hypoxic subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5571.

Effects of the application of low-temperature atmospheric plasma on titanium implants on wound healing in peri-implant connective tissue in rats

PurposeThis study aimed to clarify the effects of surface modification of titanium (Ti) implants by low-temperature atmospheric pressure plasma treatment on wound healing and cell attachment for biological sealing in peri-implant soft tissue.MethodsHydrophilization to a Ti disk using a handheld low-temperature atmospheric pressure plasma device was evaluated by a contact angle test and compared with an untreated group. In in vivo experiments, plasma-treated pure Ti implants using a handheld plasma device (experimental group: PL) and untreated implants (control group: Cont) were placed into the rat upper molar socket, and samples were harvested at 3, 7 and 14 days after surgery. Histological evaluation was performed to assess biological sealing, collagen- and cell adhesion-related gene expression by reverse transcription quantitative polymerase chain reaction, collagen fiber detection by Picrosirius Red staining, and immunohistochemistry for integrins.ResultsIn in vivo experiments, increased width of the peri-implant connective tissue (PICT) and suppression of epithelial down growth was observed in PL compared with Cont. In addition, high gene expression of types I and XII collagen at 7 days and acceleration of collagen maturation was recognized in PL. Strong immunoreaction of integrin α2, α5, and β1 was observed at the implant contact area of PICT in PL.ConclusionsThe handheld low-temperature atmospheric pressure plasma device provided hydrophilicity on the Ti surface and maintained the width of the contact area of PICT to the implant surface as a result of accelerated collagen maturation and fibroblast adhesion, compared to no plasma application.

The protective effects of Ninjin’yoeito against liver steatosis/fibrosis in a non-alcoholic steatohepatitis model mouse

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic form of non-alcoholic fatty liver disease. Liver fibrosis leads to liver cancer and cirrhosis, and drug therapy for NASH remains lacking. Ninjin’yoeito (NYT) has shown antifibrotic effects in a model of liver fibrosis without steatosis but has not been studied for NASH. Therefore, we evaluated the efficacy of NYT in mice fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a NASH model. Compared with the normal diet group, mice fed CDAHFD showed decreased body weight and increased white adipose tissue, liver weight, and triglyceride content in the liver. Furthermore, a substantial increase in the hepatic concentration of hydroxyproline, expression of α-smooth muscle actin (α-SMA), and transforming growth factor-β was observed in CDAHFD-fed mice. Masson’s trichrome and Picro-Sirius red staining revealed a remarkable increase in collagen fiber compared with the normal diet group. Compared with mice that received CDAHFD alone, those supplemented with NYT exhibited reduced hepatic triglyceride and hydroxyproline levels and α-SMA expression. Additionally, compared with the group fed CDAHFD alone, the stained liver tissues of NYT-treated mice exhibited a reduction in Masson’s trichrome- and Picro-Sirius red-positive areas. Locomotor activity was significantly reduced in the CDAHFD-fed group compared with the normal diet group. In the NYT-treated group, the CDAHFD-induced decrease in locomotor activity was significantly suppressed. The findings indicate that NYT inhibited fatty and fibrotic changes in the livers of NASH mice and alleviated the decrease in locomotor activity. Therefore, NYT may serve as a novel therapeutic approach for NASH.Graphical abstract

Recombinant Human Collagen Type III Improves Hypertrophic Scarring by Regulating the Ratio of Type I/III Collagen.

Hypertrophic scar development is a complication associated with wound healing, impacting local appearance and function. The type I/III collagen ratio affects the extent of hypertrophic scarring; a reduced ratio can ameliorate this. In this study, recombinant human collagen type III was developed. Liquid chromatography-tandem mass spectrometry was used to determine its amino acid sequence and confirm its high level of homology with natural human type III collagen. Recombinant human collagen type III displayed no cytotoxicity and did not confer skin irritation and sensitization. Immunofluorescence and western blot analyses of histidine following incubation with fibroblasts suggested cell entry of recombinant human collagen type III. Furthermore, recombinant human collagen type III promoted the synthesis of the natural type III collagen in fibroblasts, resulting in a more obvious increase of type III collagen content in fibroblasts than that of type I collagen, and then decreased the ratio of type I/III collagen. The results of 5-ethynyl-2'-deoxyuridine staining assay suggested enhanced fibroblast proliferation. Following local injection of recombinant human collagen type III, rabbit ear scarring was significantly reduced after 60 days. Vancouver Scar Scale evaluation showed that all index scores were significantly reduced. Western blotting and Picro-Sirius red staining showed that the natural type III collagen increase in scar tissue was greater than that of type I collagen, decreasing the type I/III ratio. In summary, recombinant human collagen type III can be taken up by fibroblasts and promote natural collagen synthesis-especially that of type III-thereby reducing the type I/III ratio and improving hypertrophic scarring.

Influence of systemic antibiotic therapy on the development and progression of induced apical periodontitis in Wistar rats.

The aim of this study was to investigate the influence of systemic antibiotic therapy on the development and progression of induced apical periodontitis (AP) in Wistar rats. Fifty-six rats were submitted to pulp exposure of the lower left first molar for the induction of AP. On the same day, intraperitoneal antibiotic therapy was administered once a day, for 15days, until euthanasia. The groups were formed according to the different treatments (n = 8): C-control; GEN-treated with gentamicin (10mg/Kg); AC-treated with amoxicillin (100mg/Kg); MZ-treated with metronidazole (40mg/Kg); AMP-treated with ampicillin (100mg/Kg); AMC group-treated with amoxicillin + clavulanic acid (100mg/kg); CLI-treated with clindamycin (60mg/kg). After euthanasia, the jaws were collected and processed for (1) histological and histometric analysis using hematoxylin and eosin staining, (2) analysis of collagen fibers using Picrosirius Red staining and (3) bacteriological analysis using Brown-Brenn staining. The data were analyzed statistically (p < 0.05). AP induction was confirmed in all groups. The AMC group had the lower intensity of inflammatory infiltrate (p = 0.028) and less periapical bone resorption compared to control (p = 0.006). Regarding collagen maturation, PSR staining revealed a predominance of mature collagen fibers in all groups. The AC and AMC groups had the lower amount of mature fibers and the highest amount of immature fibers, compared to all other groups (p < 0.001). All groups showed bacterial contamination; however, the AC and AMC groups showed a lower extent of bacterial contamination compared to the control (p < 0.001). It can be concluded that systemic antibiotic therapy influences the development and progression of induced AP.

Regeneration of Rabbit Calvarial Defects with Combination of Stem Cells and Enamel Matrix Derivative: A Microcomputed Tomography and Histological Evaluation Comparing Two- and Three-Dimensional Cell Constructs.

Background and Objectives: This study addresses the challenge of bone regeneration in calvarial defects, exploring the efficacy of stem cell-based therapies and enamel matrix derivative (EMD) in tissue engineering. It assesses the regenerative potential of two- and three-dimensional cell constructs combined with mesenchymal stem cells (MSCs) and EMD in rabbit calvarial defects. Materials and Methods: This research involved the use of bone-marrow-derived MSCs cultured in silicon elastomer-based concave microwells to form spheroids. White rabbits were grouped for different treatments, with Group 1 as control, Group 2 receiving only EMD, Group 3 getting EMD plus stem cells, and Group 4 being treated with EMD plus stem cell spheroids. Computed tomography (CT) and microcomputed tomography (micro-CT) imaging were used for structural assessment, while histological evaluations were conducted using hematoxylin and eosin, Masson's trichrome, and Picro-sirius red staining. Results: CT and micro-CT analyses revealed varying degrees of bone regeneration among the groups. Group 4, treated with three-dimensional MSC spheroids and EMD, showed the most significant improvement in bone regeneration. Histological analyses corroborated these findings, with Group 4 displaying enhanced bone formation and better collagen fiber organization. Conclusions: The study supported the biocompatibility and potential efficacy of three-dimensional MSC constructs combined with EMD in bone regeneration. Further investigations are needed to confirm these findings and optimize treatment protocols.

FLASH Proton Radiation Therapy Mitigates Inflammatory and Fibrotic Pathways and Preserves Cardiac Function in a Preclinical Mouse Model of Radiation-Induced Heart Disease

PurposeStudies during the past 9 years suggest that delivering radiation at dose rates exceeding 40 Gy/s, known as “FLASH” radiotherapy, enhances the therapeutic index of radiation therapy (RT) by decreasing normal tissue damage while maintaining tumor response compared to conventional (or standard) RT. This study demonstrates the cardioprotective benefits of FLASH proton RT (F-PRT) compared to standard (conventional) proton RT (S-PRT), as evidenced by reduced acute and chronic cardiac toxicities. Methods and MaterialsMice were imaged using cone beam computed tomography to precisely determine the heart's apex as the beam isocenter. Irradiation was conducted utilizing a shoot-through technique with a 5mm diameter circular collimator. Bulk RNA-sequencing was performed on non-irradiated (NR) samples, as well as apexes treated with F-PRT or S-PRT, at 2 weeks following a single 40 Gy dose. Inflammatory responses were assessed through multiplex cytokine/chemokine microbead assay and immunofluorescence analyses. Levels of perivascular fibrosis were quantified using Masson's Trichrome and Picrosirius red staining. Additionally, cardiac tissue functionality was evaluated by 2D echocardiograms at 8 and 30 weeks post-PRT. ResultsRadiation damage was specifically localized to the heart's apex. RNA profiling of cardiac tissues treated with PRT revealed that S-PRT uniquely upregulated pathways associated with DNA damage response, induction of tumor necrosis factor superfamily and inflammatory response, while F-PRT primarily affected cytoplasmic translation, mitochondrion organization and ATP synthesis. Notably, F-PRT led to a milder inflammatory response, accompanied by significantly attenuated changes in TGF-β1 and αSMA levels. Critically, F-PRT decreased collagen deposition, and better preserved cardiac functionality compared to S-PRT. ConclusionsThis study demonstrated that F-PRT reduces the induction of an inflammatory environment with lower expression of inflammatory cytokines and profibrotic factors. Importantly, the results indicate that F-PRT better preserves cardiac functionality, as confirmed by echocardiography analysis, while also mitigating the development of long-term fibrosis.

Hyaluronic Acid Hydrogel Combined With Ocimum sanctum and Tendon-Derived Extracellular Matrix Enhances Tenogenesis in Periodontal Ligament Stem Cells.

Background Ocimum sanctum (OS) is a medicinal plant with antioxidant, anti-cancer, anti-diabetic, antimicrobial, and anti-inflammatory activities. Extracellular matrix (ECM) maintains the structural stability of tissues. Hyaluronic acid (HA), which is used in hydrogel fabrication and osteochondral regeneration, increases cell viability and the expression of marker genes. Periodontal ligament stem cells (PDLSC), which are a type of mesenchymal stem cells (MSC), have self-renewing capacity and they prevent teratoma formationand promote tendon (TEN) regeneration. The aim of this study is to incorporate the phytochemical effects of Ocimum sanctuminto hyaluronic acid hydrogel scaffolds made with the MSCs in tendon ECM for increased tissue regeneration. Materials & methods Ocimum sanctum extract and methacrylated hyaluronic acid (HA-MA) were prepared. An ovine tendon sample was decellularised to obtain the ECM. The study groups of HA, TEN, HA_OS, HA_TEN, and HA_OS_TEN were prepared. The presence of tendon cells was confirmed by picrosirius red staining and the hydrogel scaffolds were analysed using scanning electron microscopy (SEM), swelling, differentiation, compression, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) compatibility analyses. Results The morphology of the samples was analysed by SEM analysis. The HA_OS_TEN sample showed the highest rate of tenogenesis, lowest swelling, high cell viability and differentiation, and optimal compression rates. Conclusion This study showed that hyaluronic acid combined with Ocimum sanctumand tendon ECM is a very good conjugation for the preparation of hydrogel scaffolds for tendon tissue regeneration using MSCs.

Fibrosis and expression of extracellular matrix proteins in human interventricular septum in aortic valve stenosis and regurgitation

Valvular heart disease leads to ventricular pressure and/or volume overload. Pressure overload leads to fibrosis, which might regress with its resolution, but the limits and details of this reverse remodeling are not known. To gain more insight into the extent and nature of cardiac fibrosis in valve disease, we analyzed needle biopsies taken from the interventricular septum of patients undergoing surgery for valve replacement focusing on the expression and distribution of major extracellular matrix protein involved in this process. Proteomic analysis performed using mass spectrometry revealed an excellent correlation between the expression of collagen type I and III, but there was little correlation with the immunohistochemical staining performed on sister sections, which included antibodies against collagen I, III, fibronectin, sarcomeric actin, and histochemistry for wheat germ agglutinin. Surprisingly, the immunofluorescence intensity did not correlate significantly with the gold standard for fibrosis quantification, which was performed using Picrosirius Red (PSR) staining, unless multiplexed on the same tissue section. There was also little correlation between the immunohistochemical markers and pressure gradient severity. It appears that at least in humans, the immunohistochemical pattern of fibrosis is not clearly correlated with standard Picrosirius Red staining on sister sections or quantitative proteomic data, possibly due to tissue heterogeneity at microscale, comorbidities, or other patient-specific factors. For precise correlation of different types of staining, multiplexing on the same section is the best approach.