METABOLIC SYNDROME-ASSOCIATED MURINE AORTIC WALL STIFFENING IS ASSOCIATED WITH PREMATURE ELASTIC FIBERS AGING.

Type 2 diabetes (T2D) constitutes a major public health problem, and despite prevention efforts, this pandemic disease is 'one of the deadliest diseases in the world. In 2022, 6.7 million T2D patients died prematurely from vascular complications. Indeed, diabetes increases the risk of myocardial infarction or stroke eightfold. The identification of the molecular actors involved in the occurrence of cardiovascular complications and their prevention are therefore major axes. Our hypothesis is that factors brought into play during physiological aging appear prematurely with diabetes progression. Our study focused on the aging of the extracellular matrix (ECM), a major element in the maintenance of vascular homeostasis. We characterized the morphological and functional aspects of aorta, with a focus on the collagen and elastic fibers of diabetic mice aged from 6 months to non-diabetic mice aged 6 months and 20 months. The comparison with the two non-diabetic models (young and old) highlighted an exacerbated activity of proteases, which could explain a disturbance in the collagen accumulation and an excessive degradation of elastic fibers. Moreover, the generation of circulating elastin-derived peptides reflects premature aging of the ECM. These extracellular elements contribute to the appearance of vascular rigidity, often the origin of pathologies such as hypertension and atherosclerosis. In conclusion, we show that diabetic mice aged 6 months present the same characteristics of ECM wear as those observed in mice aged 20 months. This accelerated aortic wall remodeling could then explain the early onset of cardiovascular diseases and, therefore, the premature death of DT2 patients.

Marker-Independent Monitoring of in vitro and in vivo Degradation of Supramolecular Polymers Applied in Cardiovascular in situ Tissue Engineering.

The equilibrium between scaffold degradation and neotissue formation, is highly essential for in situ tissue engineering. Herein, biodegradable grafts function as temporal roadmap to guide regeneration. The ability to monitor and understand the dynamics of degradation and tissue deposition in in situ cardiovascular graft materials is therefore of great value to accelerate the implementation of safe and sustainable tissue-engineered vascular grafts (TEVGs) as a substitute for conventional prosthetic grafts. In this study, we investigated the potential of Raman microspectroscopy and Raman imaging to monitor degradation kinetics of supramolecular polymers, which are employed as degradable scaffolds in in situ tissue engineering. Raman imaging was applied on in vitro degraded polymers, investigating two different polymer materials, subjected to oxidative and enzymatically-induced degradation. Furthermore, the method was transferred to analyze in vivo degradation of tissue-engineered carotid grafts after 6 and 12 months in a sheep model. Multivariate data analysis allowed to trace degradation and to compare the data from in vitro and in vivo degradation, indicating similar molecular observations in spectral signatures between implants and oxidative in vitro degradation. In vivo degradation appeared to be dominated by oxidative pathways. Furthermore, information on collagen deposition and composition could simultaneously be obtained from the same image scans. Our results demonstrate the sensitivity of Raman microspectroscopy to determine degradation stages and the assigned molecular changes non-destructively, encouraging future exploration of this techniques for time-resolved quality assessment of in situ tissue engineering processes.

Lipidome profiling with Raman microspectroscopy identifies macrophage response to surface topographies of implant materials.

Biomaterial characteristics such as surface topographies have been shown to modulate macrophage phenotypes. The standard methodologies to measure macrophage response to biomaterials are marker-based and invasive. Raman microspectroscopy (RM) is a marker-independent, noninvasive technology that allows the analysis of living cells without the need for staining or processing. In the present study, we analyzed human monocyte-derived macrophages (MDMs) using RM, revealing that macrophage activation by lipopolysaccharides (LPS), interferons (IFN), or cytokines can be identified by lipid composition, which significantly differs in M0 (resting), M1 (IFN-γ/LPS), M2a (IL-4/IL-13), and M2c (IL-10) MDMs. To identify the impact of a biomaterial on MDM phenotype and polarization, we cultured macrophages on titanium disks with varying surface topographies and analyzed the adherent MDMs with RM. We detected surface topography-induced changes in MDM biochemistry and lipid composition that were not shown by less sensitive standard methods such as cytokine expression or surface antigen analysis. Our data suggest that RM may enable a more precise classification of macrophage activation and biomaterial-macrophage interaction.

Imaging of α-Synuclein Aggregates in a Rat Model of Parkinson's Disease Using Raman Microspectroscopy.

A hallmark of Parkinson's disease (PD) is the formation of Lewy bodies in the brain. Lewy bodies are rich in the aggregated form of misfolded α-Synuclein (α-Syn). The brain from PD patients can only be analyzed after postmortem, therefore, limiting the diagnosis of PD to the manifestation of motor symptoms. In PD patients and animal models, phosphorylated α-Syn was detected in the peripheral tissues including the gut, thus, raising the hypothesis that early-stage PD could be diagnosed based on colon tissue biopsies. Non-invasive marker-free technologies represent ideal methods to potentially detect aggregated α-Syn in vivo. Raman microspectroscopy has been established for the detection of molecular changes such as alterations of protein structures. Using Raman imaging and microspectroscopy, we analyzed the olfactory bulb in the brain and the muscularis mucosae of colon tissue sections of a human BAC-SNCA transgenic (TG) rat model. Raman images from TG and WT rats were investigated using principal component analysis (PCA) and true component analysis (TCA). Spectral components indicated protein aggregates (spheroidal oligomers) in the TG rat brain and in the colon tissues even at a young age but not in WT. In summary, we have demonstrated that Raman imaging is capable of detecting α-Syn aggregates in colon tissues of a PD rat model and making it a promising tool for future use in PD pathology.

Distinct Effects of Heparin and Interleukin-4 Functionalization on Macrophage Polarization and In Situ Arterial Tissue Regeneration Using Resorbable Supramolecular Vascular Grafts in Rats.

Two of the greatest challenges for successful application of small-diameter in situ tissue-engineered vascular grafts are 1) preventing thrombus formation and 2) harnessing the inflammatory response to the graft to guide functional tissue regeneration. This study evaluates the in vivo performance of electrospun resorbable elastomeric vascular grafts, dual-functionalized with anti-thrombogenic heparin (hep) and anti-inflammatory interleukin 4 (IL-4) using a supramolecular approach. The regenerative capacity of IL-4/hep, hep-only, and bare grafts is investigated as interposition graft in the rat abdominal aorta, with follow-up at key timepoints in the healing cascade (1, 3, 7 days, and 3 months). Routine analyses are augmented with Raman microspectroscopy, in order to acquire the local molecular fingerprints of the resorbing scaffold and developing tissue. Thrombosis is found not to be a confounding factor in any of the groups. Hep-only-functionalized grafts resulted in adverse tissue remodeling, with cases of local intimal hyperplasia. This is negated with the addition of IL-4, which promoted M2 macrophage polarization and more mature neotissue formation. This study shows that with bioactive functionalization, the early inflammatory response can be modulated and affect the composition of neotissue. Nevertheless, variability between graft outcomes is observed within each group, warranting further evaluation in light of clinical translation.

Raman microspectroscopy and Raman imaging reveal biomarkers specific for thoracic aortic aneurysms.

Aortic rupture and dissection are life-threatening complications of ascending thoracic aortic aneurysms (aTAAs), and risk assessment has been largely based on the monitoring of lumen size enlargement. Temporal changes in the extracellular matrix (ECM), which has a critical impact on aortic remodeling, are not routinely evaluated, and cardiovascular biomarkers do not exist to predict aTAA formation. Here, Raman microspectroscopy and Raman imaging are used to identify spectral biomarkers specific for aTAAs in mice and humans by multivariate data analysis (MVA). Multivariate curve resolution-alternating least-squares (MCR-ALS) combined with Lasso regression reveals elastic fiber-derived (Ce1) and collagen fiber-derived (Cc6) components that are significantly increased in aTAA lesions of murine and human aortic tissues. In particular, Cc6 detects changes in amino acid residues, including phenylalanine, tyrosine, tryptophan, cysteine, aspartate, and glutamate. Ce1 and Cc6 may serve as diagnostic Raman biomarkers that detect alterations of amino acids derived from aneurysm lesions.

Nidogen-1 Mitigates Ischemia and Promotes Tissue Survival and Regeneration.

Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet ß-cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen-1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic ß-cells via the αvß3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions.

Elastin-like hydrogel stimulates angiogenesis in a severe model of critical limb ischemia (CLI): An insight into the glyco-host response.

Critical limb ischemia (CLI) is characterized by the impairment of microcirculation, necrosis and inflammation of the muscular tissue. Although the role of glycans in mediating inflammation has been reported, changes in the glycosylation following muscle ischemia remains poorly understood. Here, a murine CLI model was used to show the increase of high mannose, α-(2, 6)-sialic acid and the decrease of hybrid and bisected N-glycans as glycosylation associated with the ischemic environment. Using this model, the efficacy of an elastin-like recombinamers (ELR) hydrogel was assessed. The hydrogel modulates key angiogenic signaling pathways, resulting in capillary formation, and ECM remodeling. Arterioles formation, reduction of fibrosis and anti-inflammatory macrophage polarization wa also induced by the hydrogel administration. Modulation of glycosylation was observed, suggesting, in particular, a role for mannosylation and sialylation in the mediation of tissue repair. Our study elucidates the angiogenic potential of the ELR hydrogel for CLI applications and identifies glycosylation alterations as potential new therapeutic targets.

Non-invasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model.

The increasing prevalence of diabetes, its heterogeneity, and the limited number of treatment options drive the need for physiologically relevant assay platforms with human genetic background that have the potential to improve mechanistic understanding and e\xpedite diabetes-related research and treatment. In this study, we developed an endocrine pancreas-on-a-chip model based on a tailored microfluidic platform, which enables self-guided trapping of single human pseudo-islets. Continuous, low-shear perfusion provides a physiologically relevant microenvironment especially important for modeling and monitoring of the endocrine function as well as sufficient supply with nutrients and oxygen. Human pseudo-islets, generated from the conditionally immortalized EndoC-ßH3 cell line, were successfully injected by hydrostatic pressure-driven flow without altered viability. To track insulin secretion kinetics in response to glucose stimulation in a time-resolved manner, dynamic sampling of the supernatant as well as non-invasive real-time monitoring using Raman microspectroscopy was established on-chip. Dynamic sampling indicated a biphasic glucose-stimulated insulin response. Raman microspectroscopy allowed to trace glucose responsiveness in situ and to visualize different molecular structures such as lipids, mitochondria and nuclei. In-depth spectral analyses demonstrated a glucose stimulation-dependent, increased mitochondrial activity, and a switch in lipid composition of insulin secreting vesicles, supporting the high performance of our pancreas-on-a-chip model.

Donor age significantly influences the Raman spectroscopic biomolecular fingerprint of human pancreatic extracellular matrix proteins following collagenase-based digestion.

Despite the enormous advances in the field of clinical pancreatic islet transplantation over the past two decades, the human islet isolation procedure remains suboptimal. Islets are extracted (isolated) from the exocrine tissue of donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current isolation enzyme blends are ineffective at digesting pancreases from younger donors with low body mass indexes (BMI). However, age-related differences in pancreatic matrix digestion have not been studied in detail at the molecular level. To address this, we investigated ECM digestion in purified ECM proteins and in pancreatic tissue sections from younger (≤30 years; n = 5) and older (>55 years; n = 5) BMI matched donors, using Raman microspectroscopy (RMS). The Raman spectral profiles for purified collagens I, IV, VI and laminins were significantly altered following controlled enzyme treatment. Pancreatic cryosections were treated with Serva collagenase, NP, or the two enzymes combined, at clinically relevant concentrations. RMS demonstrated that the ECM at the islet-exocrine interface was differentially digested with respect to donor age. The action of collagenase was affected to a greater extent than NP. RMS is a powerful, marker-independent technology for characterising the human pancreatic ECM and demonstrating differences between donor types. Ongoing detailed studies using RMS will assist the development of donor-specific enzyme blends, increasing the overall success of human islet isolation and benefiting many people with type 1 diabetes worldwide. STATEMENT OF SIGNIFICANCE: Pancreatic islet transplantation is a minimally invasive treatment, which can reverse Type 1 Diabetes Mellitus (T1DM) in selected patients. Islets of Langerhans are extracted (isolated) from the exocrine tissue of human donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current enzymes are ineffective at digesting pancreases from younger donors. Using Raman microspectroscopy we demonstrate that donor age affects the enzymatic digestion of the pancreatic ECM at the molecular level. Collagenase activity is affected to a greater extent than NP. These findings will assist the development of donor-specific enzymes, thereby increasing the overall success of islet isolation and benefiting many people with T1DM worldwide.

Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging.

DNA methylation plays a critical role in the regulation of gene expression. Global DNA methylation changes occur in carcinogenesis as well as early embryonic development. However, the current methods for studying global DNA methylation levels are invasive and require sample preparation. The present study was designed to investigate the potential of Raman microspectroscopy and Raman imaging as non-invasive, marker-independent and non-destructive tools for the detection of DNA methylation in living cells. To investigate global DNA methylation changes, human colon carcinoma HCT116 cells, which were hypomorphic for DNA methyltransferase 1, therefore showing a lower global DNA methylation (DNMT1-/- cells), were compared to HCT116 wildtype cells. As a model system for early embryogenesis, murine embryonic stem cells were adapted to serum-free 2i medium, leading to a significant decrease in DNA methylation. Subsequently, 2i medium -adapted cells were compared to cells cultured in serum-containing medium. Raman microspectroscopy and imaging revealed significant differences between high- and low-methylated cell types. Higher methylated cells demonstrated higher relative intensities of Raman peaks, which can be assigned to the nucleobases and 5-methylcytosine. Principal component analysis detected distinguishable populations of high- and low-methylated samples. Based on the provided data we conclude that Raman microspectroscopy and imaging are suitable tools for the real-time, marker-independent and artefact-free investigation of the DNA methylation states in living cells.

Non-invasive functional molecular phenotyping of human smooth muscle cells utilized in cardiovascular tissue engineering.

Smooth muscle cell (SMC) diversity and plasticity are limiting factors in their characterization and application in cardiovascular tissue engineering. This work aimed to evaluate the potential of Raman microspectroscopy and Raman imaging to distinguish SMCs of different tissue origins and phenotypes. Cultured human SMCs isolated from different vascular and non-vascular tissues as well as fixed human SMC-containing tissues were analyzed. In addition, Raman spectra and images of tissue-engineered SMC constructs were acquired. Routine techniques such as qPCR, histochemistry, histological and immunocytological staining were performed for comparative gene and protein expression analysis. We identified that SMCs of different tissue origins exhibited unique spectral information that allowed a separation of all groups of origin by multivariate data analysis (MVA). We were further able to non-invasively monitor phenotypic switching in cultured SMCs and assess the impact of different culture conditions on extracellular matrix remodeling in the tissue-engineered ring constructs. Interestingly, we identified that the Raman signature of the human SMC-based ring constructs was similar to the one obtained from native aortic tissue. We conclude that Raman microspectroscopic methods are promising tools to characterize cells and define cellular and extracellular matrix components on a molecular level. In this study, in situ measurements were marker-independent, fast, and identified cellular differences that were not detectable by established routine techniques. Perspectively, Raman microspectroscopy and MVA in combination with artificial intelligence can be suitable for automated quality monitoring of (stem) cell and cell-based tissue engineering products. STATEMENT OF SIGNIFICANCE: The accessibility of autologous blood vessels for surgery is limited. Tissue engineering (TE) aims to develop functional vascular replacements; however, no commercially available TE vascular graft (TEVG) exists to date. One limiting factor is the availability of a well-characterized and safe cell source. Smooth muscle cells (SMCs) are generally used for TEVGs. To engineer a TEVG, proliferating SMCs of the synthesizing phenotype are essential, whereas functional, sustainable TEVGs require SMCs of the contractile phenotype. SMC diversity and plasticity are therefore limiting factors, also for their quality monitoring and application in TE. In this study, Raman microspectroscopy and imaging combined with machine learning tools allowed the non-destructive, marker-independent characterization of SMCs, smooth muscle tissues and TE SMC-constructs. The spectral information was specific enough to distinguish for the first time the phenotypic switching in SMCs in real-time, and monitor the impact of culture conditions on ECM remodeling in the TE SMC-constructs.

Marker-Independent In Situ Quantitative Assessment of Residual Cryoprotectants in Cardiac Tissues.

Cryomedium toxicity is a major safety concern when transplanting cryopreserved organs. Therefore, thorough removal of potentially toxic cryoprotective agents (CPAs) is required before transplantation. CPAs such as dimethyl-sulfoxide (DMSO), propylene glycol (PG), and formamide (FMD), routinely employed in ice-free cryopreservation (IFC), have advantages in long-term preservation of tissue structures compared with conventional cryopreservation employing lower CPA concentrations. This study evaluated the impact of potential residual CPAs on human cardiac valves. Raman microspectroscopy and Raman imaging were established as nondestructive marker-independent techniques for in situ quantitative assessment of CPA residues in IFC valve tissues. In detail, IFC valve leaflets and supernatants of the washing solutions were analyzed to determine the washing efficiency. A calibration model was developed according to the CPA's characteristic Raman signals to quantify DMSO, PG and FMD concentrations in the supernatants. Single point Raman measurements were performed on the intact tissues to analyze penetration properties. In addition, Raman imaging was utilized to visualize potential CPA residues. Our data showed that washing decreased the CPA concentration in the final washing solution by 99%, and no residues could be detected in the washed tissues, validating the multistep CPA removal protocol routinely used for IFC valves. Raman analysis of unwashed tissues showed different permeation characteristics depending on each CPA and their concentration. Our results demonstrate a great potential of Raman microspectroscopy and Raman imaging as marker-independent in situ tissue quality control tools with the ability to assess the presence and concentration of different chemical agents or drugs in preimplantation tissues.

Non-invasive characterization of hybrid gelatin:poly-l-lactide electrospun scaffolds using second harmonic generation and multiphoton imaging.

Hybrid scaffolds composed of synthetic polymers and naturally occurring components have become more relevant in the field of tissue engineering and regenerative medicine. Synthetic polymers are responsible for scaffold durability, strength and structural integrity; however, often do not provide biological signals. Introducing a biological component leads to more advanced and biocompatible scaffolds. In order to use these scaffolds as implants, a deeper knowledge of material characteristics and the impact of the biological component on the scaffold mechanical properties are required. Furthermore, it is necessary to implement fast, easy and non-invasive methods to determine material characteristics. In this work, we aimed to generate gelatin-poly-l-lactide (PLA) hybrids via electrospinning with defined, controllable and tunable scaffold characteristics. Using Raman microspectroscopy, we demonstrated the effectiveness of the cross-linking reaction and evaluated the increasing PLA content in the hybrid scaffolds with a non-invasive approach. Using multiphoton microscopy, we showed that gelatin fibers electrospun from a fluorinated solvent exhibit a second harmonic generation (SHG) signal typical for collagen-like structures. Compared to pure gelatin, where the SHG signal vanishes after cross-linking, the signal could be preserved in the hybrid scaffolds even after cross-linking. Furthermore, we non-invasively imaged cellular growth of human dermal fibroblasts on the hybrid electrospun scaffolds and performed fluorescence lifetime imaging microscopy on the cell-seeded hybrids, where we were able to discriminate between cells and scaffolds. Here, we successfully employed non-invasive methods to evaluate scaffold characteristics and investigate cell-material interactions.