Adipose-derived stromal vascular fraction therapy improves age-related adrenergic mediated microvascular dysfunction and increases revascularization potential following injury.

The role of the microcirculation is to balance blood flow and oxygen delivery to meet local metabolic and oxygen demands. With advancing age, the health of the endothelium declines leading to improper augmentation of the microcirculation; decreasing vasoreactivity and angiogenic potential which can further increase the risk of ischemia. The multiple contributing factors that drive the decline in endothelial health with age make traditional pharmacological interventions challenging whereas cell-based therapies can exert multifactorial gains. Adipose-derived stromal vascular fraction (SVF) is an emerging therapeutic for its easily accessible, autologous, anti-inflammatory, and angiogenic properties. It is a heterogenous population comprised of leukocyte and vascular cell populations as well as a small population of mesenchymal stem cells. Coronary perfusion and isolated coronary microvessels from young, old, and old treated with young SVF, female Fisher-344 rats were examined for health and functionality to beta-adrenergic receptor agonists (b-AR). Advancing age decreases coronary perfusion and vasoreactivity to b1-AR agonist norepinephrine (NE). A one-time tail vein injection of 10 million SVF cells 4 weeks prior reversed the age-related impairment in coronary microvascular dysfunction. Furthermore, isolated coronary microvessels from young, old, and old treated with SVF rats were examined for age- and SVF-related alterations in b1-AR cell signaling. Advancing age reduced the expression of b1-ARs on coronary microvessels and increased the expression of vasodilatory inhibitors, GRK2 and Gai. While SVF fully restored the b1-AR population it only marginally mitigated the inhibitor expression back to young control levels. Advancing age altered the cellular composition of SVF promoting a more pro-inflammatory phenotype with increased M1 macrophages and various types of T cells with a reduced mesenchymal stem cell population. Intravenously injected SVF can disseminate and engraft into the microcirculation of mesenteric windows; connective tissues located along the small intestines. SVF from young donors can significantly increase vascularized area of an aged mesenteric window subjected to a hypoxic-like injury compared to aged SVF which only marginally improves vascular area. Advancing age can drive a decline in the functionality of the microcirculation. SVF therapy offers a promising vascular therapeutic reversing the age-related dysfunction, increasing organ perfusion, and promoting revascularization following injury

Cyclic Nucleotide Signaling and the Cardiovascular System

The cyclic nucleotides 3',5'-adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) play important roles in the control of cardiovascular function under physiological and pathological conditions. In this book, which is a reprint of a Special Issue of the Journal of Cardiovascular Development and Disease entitled "Cyclic Nucleotide Signaling and the Cardiovascular System", internationally recognized experts give an overview of this vibrant scientific field. The first series of articles deal with the localization and function of membrane-bound and soluble adenylate cyclases, followed by articles on the roles of phosphodiesterase isoforms in the heart. Cyclic nucleotide signaling takes place in nanodomains and the A-kinase anchor proteins (AKAPS) are essential for the compartmentalized assembly of signaling proteins into functional complexes. Reviews on the role of AKAP proteins in the physiology and pathophysiology of the heart are also included in this book. Cyclic nucleotides act through effector proteins and articles on EPAC and POPDC proteins inform the reader of recent developments on these topics. A major advancement in our understanding of cyclic nucleotide signaling came through the use of genetically encoded cAMP sensor molecules, and a series of articles review the current insight that these reporter molecules have provided. The final set of articles in this book deals with the association of the cyclic nucleotide pathway and cardiovascular disease as well as the development of novel therapeutic approaches. Thomas Brand and Enno Klussmann Special Issue Editor

Evaluation of the Role of Microvascular Pathology on Peripheral Artery Disease

Background: Peripheral Artery Disease (PAD) begins with atherosclerotic narrowing of arteries, including those that supply the legs. Individuals with PAD experience pain during walking, which becomes increasingly limiting. Studies from our group and others have shown that a myopathy is present in the skeletal muscle of PAD patients, and is characterized by myofiber degeneration, fibrosis, and remodeling of vessels ranging from 50 – 150 mm in diameter. However, microvascular pathology, particularly of the smallest microvessels (5 – 15 mm in diameter) remains poorly characterized. Furthermore, little is known about the relationships between microvascular architecture, microperfusion, and patient walking performance. We hypothesize that microvascular pathology is present in the terminal microvasculature of PAD muscle compared to control and worsens with PAD severity. Additionally, we hypothesize that microvascular architecture is associated with deficits in micro- and macro- perfusion and walking performance in PAD patients with intermittent claudication (IC). Methods: Gastrocnemius biopsy specimens were collected from control, PAD patients with IC, and PAD patients with critical limb ischemia. Microvascular architecture, microvascular fibrosis, total collagen, and the abundance and phenotype of pericytes were quantified. Microvascular perfusion was assessed by Contrast Enhanced Ultrasonography (CEU). Gardner walking protocols were used to assess claudication onset time (COT) and peak walking time (PWT). Patients also completed the Walking Impairment Questionnaire (WIQ). Results: Microvascular pathology increased with advancing PAD severity and included progressive increases in basement membrane thickening, abundance of aSMA+ pericytes, and microvessel density. In advanced PAD muscle, increases were observed in total fibrotic burden and peri-microvascular Collagen I and IV deposition. aSMA+ pericytes expressed TGF-b1. Relationships were observed between microvascular architecture and microperfusion both at rest and after ischemic stress. Microvascular architecture was associated with macrovascular hemodynamic restrictions. Microvascular architecture was associated with COT, PWT, and patient self-reports of walking speed, walking distance, and stair climbing ability. Conclusions: Microvascular pathology worsens with PAD severity in association with fibrosis. Alteration of microvascular architecture contributes to microperfusion deficits and walking limitations in PAD

Molecular mechanisms of vascular remodeling in pulmonary arterial hypertension : the implication of tyrosine kinase inhibitors and epigenetic events in the disease

L'hypertension artérielle pulmonaire (HTAP) est une maladie rare caractérisée par une obstruction progressive et un remodelage vasculaire des artères pulmonaires distales, conduisant à une pression artérielle pulmonaire moyenne supérieure à 20mmHg. L'augmentation de la pression aboutit à une dysfonction du ventricule droit et la mort. À l'heure actuelle, il n'existe pas de traitement curatif pour l'HTAP. Il est donc primordial d'identifier de nouvelles cibles thérapeutiques. Comme dans le cancer, les cellules musculaires lisses de l'artère pulmonaire des patients atteints d'HTAP présentent un phénotype hyper-prolifératif et résistant à l'apoptose, entraînant un remodelage vasculaire pulmonaire. Ainsi, plusieurs stratégies thérapeutiques anti cancéreuses pourraient être utiles pour le traitement de l'HTAP. Compte tenu de l'expression altérée des récepteurs tyrosines kinases dans l'HTAP ainsi que dans le cancer, les inhibiteurs de tyrosine-kinases (ITK) ont été mise sur le marché pour le traitement de différents types de cancers et ont été envisagées dans le cadre de l'HTAP. Ainsi, l'ITK, Imatinib, a été capable de régresser l'HTAP induite dans des modèles expérimentaux, tandis que l'administration d'un autre inhibiteur, le Dasatinib, était associée au développement de l'HTAP. Récemment, plusieurs études observationnelles ont démontré le développement de l'HTAP chez des patients atteints d'un cancer du poumon non à petites cellules (CPNPC) présentant des réarrangements de la kinase lymphocytaire anaplasique (ALK) et qui ont reçu des ITK ALK/cMET, notamment Xalkori (R-Crizotinib), Ceritinib, Brigatinib et Lorlatinib. Étant donné que l'hypertension pulmonaire peut être associée à plusieurs maladies, y compris le cancer du poumon, la question demeure de savoir si le développement de l'HTAP chez les patients atteints d'un cancer du poumon recevant l'ITK ALK/c-MET représente un événement indésirable du médicament ou un résultat de la propagation de la maladie. Ainsi, l'objectif principal de chapitre 1 est de déterminer si le R-Crizotinib (connu sous le nom de Xalkori et qui est une première ligne de traitement des patients ayant un CPNPC-ALK positif) exacerbe l'HTAP et/ou prédispose à l'HTAP dans des modèles animaux. In vivo, le traitement par R-Crizotinib a entraîné une élévation marquée de la pression systolique du ventricule droit et de la pression artérielle pulmonaire moyenne associée à une augmentation de l'épaisseur de la paroi médiale des artères pulmonaires distales. De plus, R-Crizotinib, administré avant l'exposition à une faible dose de monocrotaline (MCT), induit une réponse hypertensive pulmonaire exagérée, comme en témoigne une augmentation de la pression systolique du ventricule droit et de la pression artérielle pulmonaire moyenne, de l'épaisseur de la paroi médiale et une diminution du débit cardiaque. In vitro, nous avons démontré que le traitement avec R-Crizotinib réduit la prolifération des cellules endothéliales contrôles de l'artère pulmonaire, effet associé à la formation de cellules multinucléées, ce qui est généralement observée dans les cellules qui meurent d'une une catastrophe mitotique. En conclusion, nous avons démontré que l'agent anticancéreux R-Crizotinib favorise le dysfonctionnement des cellules endothéliales, conduisant à la prédisposition et à l'exacerbation de l'HTAP dans des modèles animaux. Les dernières années de recherche ont approuvé l'importance des marques épigénétiques dans le développement de l'HTAP. En effet, nous nous sommes intéressés, dans le deuxième volet de la thèse, au facteur épigénétique « G9a », qui s'est révélé surexprimé dans différents types de cancers, favorisant la survie et la prolifération des cellules. Compte tenu de l'analogie cancer/HTAP, G9a fut un candidat idéal pour l'étude de son potentiel rôle dans le développement de l'HTAP. Ainsi, l'objectif principal du chapitre 2 est de déterminer si G9a est impliqué dans la progression et la pathogenèse de l'HTAP et de déterminer si son inhibition est bénéfique dans les modèles animaux. Nous avons démontré que G9a est surexprimé dans les artères pulmonaires de patients HTAP et dans les modèles expérimentaux. In vitro, l'inhibition pharmacologique de G9a à l'aide de BIX01294 diminue drastiquement la capacité d'hyper prolifération et la résistance à l'apoptose des cellules musculaires lisses HTAP. Grâce au séquençage d'ARN, nous avons démontré que l'inhibition de G9a s'accompagnait d'une altération du flux d'autophagie et d'une accumulation de lipides. Enfin, le traitement thérapeutique avec BIX01294 a réduit le remodelage vasculaire pulmonaire et la pression artérielle pulmonaire moyenne dans un modèle expérimentale de rat et a également amélioré l'hémodynamique pulmonaire et la fonction ventriculaire droite dans un autre modèle de souris. Ces résultats suggèrent que l'inhibition de G9a pourrait représenter une nouvelle approche thérapeutique dans l'HTAP.Pulmonary arterial hypertension (PAH) is a rare and fatal disease characterized by "a progressive loss and obstructive remodeling of pulmonary arteries (PAs) leading to a mean pulmonary arterial pressure (mPAP) greater than 20mmHg. The persistent elevation of pulmonary pressures leads to right ventricular dysfunction and death. Currently, there is no cure for patients with PAH, which increases the need to develop new and effective therapeutic strategies. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients exhibit a "cancer-like" hyperproliferative and apoptosis-resistant phenotype leading to pulmonary vascular remodeling. Therefore, several anti-cancer therapies could be useful for the treatment of PAH. Given the altered expression of receptor tyrosine kinases and their ligands in PAH as well as in cancer, tyrosine kinase inhibitors (TKIs) have been marketed for the treatment of different types of cancers and have been in the spotlight for anti-PAH drug research. Indeed, the tyrosine kinase inhibitor, Imatinib, was able to regress established PAH in experimental models, while the administration of another inhibitor, Dasatinib, was associated with the development of PAH. Recently, several observational studies have highlighted the development of PAH in patients with non-small cell lung cancer (NSCLC) with anaplastic lymphocyte kinase (ALK) rearrangements who received ALK/cMET TKIs, including Xalkori (R-crizotinib), Ceritinib, Brigatinib, and Lorlatinib. Since pulmonary hypertension can be associated with several diseases, including lung cancer, the question remains whether the development of PAH in lung cancer patients receiving cMET/ALK TKIs represents an adverse drug event or a result of disease spread. Thus, the main objective of Chapter 1 is to determine whether R-Crizotinib (known as Xalkori and which is the first-line treatment for patients with advanced ALK-positive NSCLC) exacerbates PAH and/or predisposes to PAH in experimental animal models. In vivo, R-Crizotinib treatment resulted in a marked elevation of the right ventricular systolic pressure (RVSP) and mPAP which was associated with an increased medial wall thickness of the distal PAs. Additionally, we found that pretreatment of rats with R-Crizotinib, induced an exaggerated pulmonary hypertensive response, as evidenced by the increased RVSP, mPAP, medial wall thickness, and decreased cardiac output. In vitro, we have demonstrated that treatment with R-Crizotinib reduces the proliferation of control pulmonary artery endothelial cells, which was accompanied by the appearance of multinucleated cells, a feature commonly seen in cells dying from mitotic catastrophe. In conclusion, we have demonstrated for the first time that the anticancer agent R-Crizotinib promotes endothelial cell dysfunction, leading to susceptibility and exacerbation of PAH in animal models. Previous studies have demonstrated the importance of epigenetic marks in the development and progression of PAH. Indeed, we were interested in the second part of the thesis, in the epigenetic factor "G9a", which was found to be overexpressed in different types of cancers, promoting cell survival and proliferation. Given the similarities between PAH and cancer, G9a was the ideal candidate to study in PAH. Thus, the main objective of Chapter 2 is to determine if G9a is involved in the progression and pathogenesis of PAH and to determine if its inhibition is beneficial in PAH animal models. We demonstrated that G9a is overexpressed in PAs of PAH patients and in experimental models. In vitro, we found that pharmacological inhibition of G9a using BIX01294 drastically reduces the PAH-PASMC proliferation and survival. Through RNA sequencing analysis, we demonstrated that G9a inhibition is accompanied by an impaired autophagy flux and lipid accumulation. Finally, therapeutic treatment with BIX01294 reduced pulmonary vascular remodeling as well as mPAP in an experimental rat model and also improved pulmonary hemodynamics and right ventricular function in another PAH mouse model. These results suggest that G9a inhibition could represent a new therapeutic approach in PAH

Targeting hepatic stellate cells to prevent or reverse liver fibrosis

Activation of stellate cells as a target for the treatment of liver fibrosisIn his thesis, Zhang introduces the crucial role of hepatic stellate cell activation in the development of liver fibrosis. Activated liver stellate cells are the main precursors of myofibroblasts. Therapy targeting hepatic stellate cells can prevent or reverse liver fibrosis.The aging ("senescence") of hepatic stellate cells is interpreted as a mechanism that protects against the progression of liver fibrosis. The biomarkers, signaling pathways and likely effects of hepatic stellate cell senescence are explained. Zhang proposes that the biomarkers P21 (cell cycle arrest), senescence-associated β-galactosidase (lysosomal galactosidase), and interleukin-6 (senescence-associated secretory phenotype) can be used to identify senescent hepatic stellate cells. Although the senescence of liver stellate cells has not yet been fully elucidated, therapy-induced senescence of hepatic stellate cells, followed by senolytics, may be an optimal strategy for combating liver fibrosis.Esculetin, a coumarin derivative, has also been shown to cause the senescence of hepatic stellate cells. It appears that in senescent hepatic stellate cells, collagen production and cell proliferation are reduced. In addition, senescent hepatic stellate cells develop resistance to a return to active proliferation

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Investigating the Mechanisms of Hepatocyte or Biliary Epithelial Cell-Mediated Liver Regeneration

The liver performs a wide array of functions such that it is indispensable for survival. Uniquely, the liver is the only human internal organ capable of regeneration. Failure of liver regeneration is thought to induce progression of chronic liver disease (CLD) to cirrhosis and end-stage liver disease, which is currently the 12th leading cause of death in the U.S. The only current treatment for end-stage liver disease is a liver transplant, for which there is a major dearth of donor organs. Therefore, understanding the molecular mechanisms underlying liver regeneration could lead to the development of desperately needed new therapies for liver disease. There are two main epithelial cell types in the liver: hepatocytes and biliary epithelial cells (BECs). Typically, liver regeneration after an acute injury is mediated by proliferation of hepatocytes. This is the case after partial hepatectomy (PHx), where 2/3 of the liver is surgically removed. We identified a role for bromodomain and extraterminal (BET) proteins in driving hepatocyte proliferation after PHx. BET proteins are a family of chromatin readers that interact with the basic transcriptional machinery to promote expression of virtually all genes. One pathway that we found to be especially sensitive to BET protein inhibition post-PHx was the Wnt/β-catenin signaling pathway, which is an important pathway in both liver development and multiple models of liver regeneration. One of these models involves liver regeneration when hepatocyte proliferation is impaired, a model in which BECs are theorized to differentiate into hepatocytes to mediate liver regeneration. We demonstrated that mice which lack liver-specific Wnt/β-catenin signaling (KO mice) exposed to choline-deficient, ethionine-supplemented (CDE) diet-induced liver injury have impaired hepatocyte proliferation. Furthermore, we utilized genetic lineage tracing of both hepatocytes and BECs to prove that BECs differentiate into hepatocytes in KO mice but not their wild-type counterparts following CDE diet-induced liver injury and recovery. Overall, our work has elucidated important signaling pathways driving liver regeneration in multiple models of liver injury and sets the stage for future work to identify clinically-relevant treatments which can enhance hepatic repair