Current treatments for peripheral arterial occlusive disease (PAOD) have limited success, so there is a need to develop more effective treatments. Because patients with native collaterals have a better prognosis, promoting collateral arteriogenesis is a potential PAOD treatment. Additionally, female PAOD patients have a worse response to treatment and a worse prognosis compared to males, which could be due to impaired collateralization. Cell transplantation is a potential treatment option to promote collateral arteriogenesis. Bone marrow derived stem cells are the main cell type that has been investigated, but they have had limited clinical success. Delivering a stem cell type native to the tissue like myogenic stem cells could have improved outcomes. In this study, the lateral spinotrapezius feed artery was ligated in male and female Balb/C mice to induce collateral capillary arteriogenesis, and 7 days post ligation ­­­­arterialized collateral capillary (ACC) number and diameter were determined. There were no differences between sexes, which could be because young, healthy mice were used in this study rather than aged and diseased models. Because we observed no sex differences, we then assessed the effect of myogenic cell transplantation in male mice only. Immediately following ligation of the spinotrapezius feed artery, mice were treated with myogenic cells, thrombin, or vehicle, and 7 days post ligation ACC number and diameter were determined. Thrombin increased ACC number, but myogenic cells had no effect. However, myogenic cells increased ACC diameter, and both myogenic cells and thrombin decreased ACC number in the region of the muscle with the largest collateral, and increased the maximum ACC diameter. Another factor that could affect ACC formation is a pre-existing collateral (PEC), which only some Balb/C mice have, so we also separated mice into PEC and non-PEC groups for analysis. In mice with a PEC, thrombin increased ACC number, and both myogenic cells and thrombin increased ACC diameter. There was a trend toward smaller arterialized capillaries in mice with a PEC, which could be because the majority of the blood flow is redirected through the PEC, so the PEC was the main vessel to remodel. These results are consistent with previous studies that indicated that thrombin augments arteriogenesis as well as increasing V-CAM, and suggest that myogenic cells have a similar effect possibly by secreting arteriogenic factors such as VEGF and MMPs. Because myogenic cells increase arteriogenesis, and macrophages are an essential regulator of arteriogenesis, we also tested the hypothesis that myogenic cells would increase macrophage content. Macrophage number increased with ligation, but there was no difference in macrophage number between any of the treatment groups. The lack of difference in macrophage number could be because the day 7 timepoint was too late, as macrophage content peaks at day 3. Because myoblasts increased arteriogenesis, they also may have increased the number M2 macrophages, which are the main macrophage contributor to arteriogenesis, but we used a general macrophage marker and could not detect an increase in M2 macrophages. In future studies, to determine if there is an increase in M2 macrophages a stain specific to M2 macrophages like CD206 could be added. Additionally, a diabetic Balb/C strain could be used to determine if arteriogenesis is impaired in males compared to females in a diseased model.

Clinical evidence indicates a higher incidence of peripheral arterial occlusive disease and associated likelihood of critical limb ischemia in women, as well as worse prognosis and decreased survival post myocardial infarction. Therefore, understanding the possible differences in underlying vascular compensation mechanisms is crucial. With arterial occlusions, necrosis and tissue injury can be naturally mitigated by the collateral circulation, improving patient prognosis. Previous sex-comparison studies describing differences in vascular remodeling are inconsistent. Therefore, the aim of this study was to describe the effect of arterial occlusion on collateral remodeling in healthy male and healthy reproductive-stage female mice. At 7 days following femoral artery ligation in C57Bl/6 and BALB/c mice, there were no sex-related differences in functional ambulatory recovery. There were no sex-related differences in mechanoadaption indicators in the collateral stem- vascular smooth muscle cell (VSMC) length and overlap, with the exception of longer smooth muscle cells in male C57Bl/6 mice, VSMC lengths 329 ± 19 verses 288 ± 13 μm, male and female. Collateral midzone luminal and abluminal diameters, as well as wall thicknesses were not different between sexes. As comprehensive sex-specific differences were not captured in our specific investigation of arteriogenesis, an evaluation of microvascular remodeling in the ischemic zone and collateral vasodilation would be of interest, as would evaluating arteriogenesis following oophorectomy with estrogen depletion. The determination of any underlying mechanistic sex-specific differences could be the foundation for which targeted therapeutics are developed, which will be crucial for closing the prognosis gap between men and women in the global treatment of peripheral arterial occlusive disease.

Peripheral artery disease (PAD) is characterized by the development of atherosclerotic plaques on arterial walls, leading to the narrowing of blood vessels, resulting in ischemia in the downstream tissue. In the United States, 12% of the adult population is affected by PAD and its related symptoms. Current surgical revascularization techniques can be effective in part of the patient population, but there is a need for other options. Alternatively, collateral blood vessels, or natural bypass arteries, enlarge to increase blood flow to the ischemic tissue in a process called arteriogenesis, which has been studied as a therapeutic option. Cell-based therapies, such as BM-MNCs, have been investigated as means to enhance arteriogenesis, but have largely failed in clinical trials. An alternative cell-based therapy candidate are myoblasts, or muscle progenitor cells. Myoblasts increase arteriogenesis in murine models and are known to interact with macrophages, which are immune cells that are primary regulators of arteriogenesis. Macrophages can polarize to inflammatory (M1) and regenerative (M2) phenotypes, with the M2 phenotype promoting enhanced arteriogenesis. This interaction suggests that myoblasts may be signaling macrophage polarization to enhance arteriogenesis. The purpose of this study was to determine if myoblasts in vitro can affect macrophage polarization into inflammatory (M1) or regenerative (M2) phenotypes. Protocols for macrophage culture and polarization were implemented, and then macrophages were co-cultured with myoblasts for 24 hours to assess the effects in vitro. Concentrations of known inflammatory (TNF-a) and regenerative (IL-10) cytokines released by macrophages were measured after co-culture with myoblasts. Surprisingly, macrophages co-cultured with myoblasts showed a decrease in both TNF-a and IL-10 compared to macrophages cultured alone. Morphology changes of macrophages were also measured after co-culture, with, surprisingly, little difference in the groups co-cultured with myoblasts. Pilot experiments suggest there may be an initial lag time greater than 24 hours for myoblasts to affect macrophage phenotype. Future work ideally will include longer time points and optimizing viability and proliferation of myoblasts in co-culture settings.

Peripheral arterial occlusive disease (PAOD) is an ischemic disease characterized by narrowing of the peripheral arteries due to the accumulation of atherosclerotic plaque in the inner lining of the vessels, which disrupts blood flow to downstream tissues. Blood can be redirected into collateral vessels, natural bypasses around arterial occlusions, causing shear-induced outward remodeling of the vessels. The enlarged vessels facilitate transfer of increased blood flow to downstream tissues. The remodeling process, however, may impair vasodilation, which in turn may cause or contribute to intermittent claudication- transient pain brought on by locomotion. To stimulate the growth of collateral arteries, the femoral arteries of young, otherwise healthy mice were ligated distally to the profunda femoris, the stem to the gracilis collateral circuit. The diameter of the profunda femoris artery was measured at rest and following gracilis muscle contraction 7 and 28 days post-surgery using intravital microscopy. Enlarged resting diameter, consistent with collateral enlargement, and impaired vasodilation was observed at day 7, but not at day 28. To determine if impaired functional vasodilation is due to impaired endothelial- or smooth muscle-dependent responses during outward remodeling, cell-dependent vasodilators were applied to the hindlimb. Endothelial- and smooth muscle-dependent vasodilation was significantly impaired 7 days post-ligation, but not 28 days after. This data supports the hypothesis that smooth muscle dysfunction causes impaired functional vasodilation in the early stages of collateral enlargement.

Functional hyperemia and arteriolar vasodilation are impaired with chronic ischemia. We sought to examine the impact of chronic ischemia on collateral artery function. For this we used two hindlimb ischemia models to dissect the impact of different repair processes on collateral function. Ligation of the femoral artery increases shear stress in the muscular branch and results in outward remodeling and arteriogenesis. In contrast, resection of the femoral artery proximal to the muscular branch induces blood flow divergence and neutral remodeling along with expectedly greater hypoxia and inflammation. On day 14 after each surgery the diameter of the muscular branch was measured using sidestream dark field (SDF) imaging before and after gracilis muscle stimulation. A slight, but not statistically significant, impairment in functional vasodilation was observed in ligated mice (69±10% average diameter increase compared to 74±7% average diameter increase). Resected mice exhibited slightly (not statistically significant) enhanced collateral artery functional vasodilation (104±16% average diameter increase) but were also refractory to the restoration of resting vascular tone following the cessation of stimulation. Outward remodeling did not significantly impair vascular function, whereas neutral remodeling and tissue hypoxia induced impaired vascular tone.

No description available.

Stroke is a leading cause of morbidity and long-term neurological disability in the U.S. Ischemic stroke, which accounts for approximately 90% of all strokes, is the result of an occlusion in the arteriole cerebrovascular network. No effective treatment options exist to provide neuroprotection from occlusion, and limited success has been seen clinically when attempting to restore blood flow to vulnerable neural tissue regions. Enhancement of pial collateral remodeling (Arteriogenesis) has recently been shown to improve blood flow and mitigate neural tissue damage following stroke (1-3). Arteriogenesis is the remodeling of pre-existing arteriole vessel which are able to re-route blood to blood-deprived regions of tissue. Arteriogenesis requires endothelial cell (EC) and smooth muscle cell proliferation, extracellular matrix degradation and recruitment of circulating bone marrow-derived cells (4-6). Unlike spouting angiogenesis, which requires weeks following occlusion to develop, arteriogenesis begins as early as 24-48hrs post-stroke (7, 8) and can expeditiously enhance blood flow to ischemic regions, making it an attractive target for therapeutic intervention. Our preliminary studies, in an EphA4 global knockout mouse model, indicated that EphA4 receptor tyrosine kinase severely limits pial arteriole collateral formation. The preliminary work also showed that activation of EC EphA4 receptor in vitro inhibited vascular formation. Additionally, ECs lining the collateral vessel have been shown to play a role in collateral remodeling (9). Taken together, the objective of this dissertation was to elucidate the cell autonomous role of the EphA4 receptor and given the central role of the EC in collateral remodeling, we postulated that EphA4 receptor on ECs the limits pial collateral formations. Using a cell-specific loss-of-function approach, we tested the hypothesis that EC-specific EphA4 plays an important role in pial collateral development and remodeling after induced stroke. The results from this dissertation show that (1) EphA4 expression on ECs suppress the formation of pial collaterals during development and limits EC growth via suppression of p-Akt in vitro (2) EC-specific EphA4 ablation leads to increased collateral remodeling, enhanced blood flow recovery, tissue protection and improved neurological behavioral outcomes after stroke and (3) Mechanistically, EphA4 limits pial collateral remodeling via attenuation of the Tie2/Angiopoietin-2 signaling pathway. The work presented in this dissertation demonstrate that EphA4 can be targeted therapeutically to increase pial collateral remodeling to alleviate neurological deficits after ischemic stroke. / Doctor of Philosophy / Stroke is the fifth leading cause of death in the United States. Ischemic stroke is the most common type of stroke and occurs when blood flow to part of the brain is impeded. Lack of blood results in cell death and tissue damage in the brain. In an effort to restore blood flow, specialized blood vessels in the brain called collaterals remodel and become larger to allow re-routed blood to the blood-deprived region of the brain. The duration it takes to remodel these remarkable blood vessels and re-route blood varies in humans, and sometimes is not able to prevent adequate tissue damage. The current work explores novel therapeutic targets to accelerate collateral remodeling in an effort to reduce tissue loss after stroke. We present studies which show that a protein called EphA4, found on endothelial cells restricts remodeling, and when inhibited in the brain can increase collateral remodeling and reduced adverse effects after ischemic stroke.

Postnatal vascular growth is a complex process involving multiple cells types whose functionality is orchestrated by a variety of soluble extracellular growth factors, mechanical stimuli, and matrix derived cues. The central goal for this dissertation project was to elucidate the role of osteopontin, a non-collagenous extracellular matrix protein, in postnatal vascular growth. At the onset, we concluded that the current methods for measurement of vascularity in small animal models were lacking. To address this shortcoming, we pursued micro-CT imaging for analysis of three-dimensional blood vessel architecture. We were able to demonstrate that micro-CT imaging provides an objective, quantitative, and three-dimensional methodology for evaluation of vascular networks that has broad applicability to preclinical studies. Next, we sought to apply the developed imaging techniques, along with other complementary methodologies, to explore the role of osteopontin in postnatal vascular growth. Osteopontin was previously known to elicit survival, migration, and other relevant activities in multiple cell types involved in postnatal vascular growth. Therefore, we sought to determine the in vivo significance of osteopontin in this process. To do so, we compared wild type and Osteopontin-/- mice for (1) their ability to form collateral vessels and functionally recover following acute induction of hind limb ischemia and (2) their capacity for neovascularization, mineralization, remodeling, and the restoration of mechanical properties during fracture healing. Data suggested that OPN is a critical regulator of collateral vessel formation and that this effect is driven by its role in mediating monocyte/macrophage migration and functionality. Secondly, we found that the presence of osteopontin was essential for normal early callus formation, neovascularization, late stage callus remodeling, and restoration of biomechanical strength. Abnormal collagen organization was observed within the remodeling fractures of Osteopontin-/- mice, and we hypothesize that a unifying link between the vascular and bone defects may be related to deficient matrix organization and remodeling. In conclusion, the imaging techniques developed in this thesis provide a novel methodology for quantitative analysis of vascular structures in small animal models. Secondly, this project has yielded an improved understanding of the basic pathophysiological mechanisms that control postnatal blood vessel growth and bone fracture healing.

Η αγγειογένεση είναι η βασική διαδικασία κατά την οποία τα αγγεία αναπτύσσονται από ήδη υπάρχοντα. Αποτελεί βασική προϋπόθεση για ένα μεγάλο αριθμό φυσιολογικών και παθολογικών διαδικασιών. Πρόσφατα καθιερώθηκε ο όρος αρτηριογένεση εστιάζοντας στην διαδικασία με την οποία ένα προϋπάρχον μικρό αρτηριόλιο ωριμάζει και αναδιαμορφώνεται σε ένα μεγαλύτερο αρτηρίδιο που μπορεί να άγει πολύ μεγαλύτερη ποσότητα αίματος στη μονάδα του χρόνου ως απάντηση στην αυξημένη ενδαγγειακή ροή και πίεση αίματος. Έτσι, η ενεργοποίηση της αρτηριογένεσης και η αιμοδυναμική υποβοήθηση των υπαρχόντων παραπλεύρων αγγείων (θεραπευτική αγγειογένεση) αποτελεί σήμερα έναν ελκυστικό ερευνητικό στόχο με σκοπό την επαναιμάτωση και συνεπώς σωτηρία ευαίσθητων στην ισχαιμία ιστών όπως η καρδιά, ο εγκέφαλος και τα άκρα. O τομέας της θεραπευτικής αγγειογένεσης και αρτηριογένεσης αποτελεί σήμερα ένα ταχέως εξελισσόμενο πεδίο στην σύγχρονη έρευνα του καρδιαγγειακού συστήματος. Η στεφανιαία και περιφερική αρτηριακή αθηροσκλήρωση αποτελεί παγκοσμίως την πρωτεύουσα καρδιαγγειακή αιτία θνησιμότητας και νοσηρότητας. Σε πολλούς ασθενείς δεν είναι εφικτή ούτε η διαδερμική ενδοαγγειακή αντιμετώπιση με αγγειοπλαστική και μεταλλικές ενδοπροθέσεις (stents) ούτε η χειρουργική επαναγγείωση του ισχαιμούντος ιστού μέσω αρτηριακής παράκαμψης (bypass). Έτσι η έρευνα για τα καρδιαγγειακά νοσήματα έχει εστιάσει την προσοχή της σε νέους ελάχιστα επεμβατικούς τρόπους επαναιμάτωσης, όπως είναι η θεραπευτική αγγειογένεση με ενδοαρτηριακή, ενδομυϊκή ή περιαγγειακή έγχυση διαφόρων αγγειογενετικών παραγόντων οι οποίοι διεγείρουν τα ενδογενή αγγειογενετικά βιοχημικά μονοπάτια και προάγουν την ανάπτυξη παράπλευρου δικτύου (αρτηριογένεση) ικανού να αναστρέψει την ιστική ισχαιμία και υποξία. Διάφοροι αγγειογενετικοί παράγοντες έχουν δοκιμαστεί στην κλινική πράξη για την ενίσχυση του παράπλευρου αρτηριδιακού δικτύου στην στεφανιαία νόσο και στην περιφερική αγγειοπάθεια με αμφιλεγόμενα μέχρι τώρα αποτελέσματα. Η θρομβίνη είναι ένα ένζυμο με δραστικότητα σερινοπρωτεάσης που αποτελεί μόριο κλειδί στους βιοχημικούς καταρράκτες της πήξης και αιμόστασης. Πρόσφατες έρευνες έχουν αποδείξει επίσης ότι η θρομβίνη ενεργοποιεί την διαδικασία της αγγειογένεσης μέσω μηχανισμών εξαρτώμενων και ανεξάρτητων από την πήξη του αίματος. Πρόσφατα βρέθηκε ότι η θρομβίνη συμμετέχει στις διεργασίες επιβίωσης των ενδοθηλιακών κυττάρων. Αυτό μπορεί να ανοίξει νέους ορίζοντες όσον αφορά το γενικότερο ρόλο της θρομβίνης στην αγγειοπροστασία και τη συμβολή της στη διατήρηση της ακεραιότητας του τοιχώματος των αγγείων. Η αλληλεπίδραση των αγγειογενετικών αυξητικών παράγοντων και των διαφόρων μηχανισμών αγγειοπροστασίας είναι ουσιώδης τόσο για την ανάπτυξη όσο και για την ωρίμανση των νέων αιμοφόρων αγγείων κατά την εξέλιξη των φαινομένων της αγγειογένεσης και της αρτηριογένεσης. Σκοπός της συγκεκριμένης ερευνητικής δουλειάς ήταν η δοκιμασία, επιβεβαίωση, και κατά κύριο λόγο η σύγκριση της αγγειογενετικής δραστηριότητας της θρομβίνης με άλλους καταξιωμένους αυξητικούς παράγοντες σε ένα in vivo μοντέλο ισχαιμίας κάτω άκρων, με απώτερο στόχο την εφαρμογή της για την πρόκληση θεραπευτικής αγγειογένεσης και επαναιμάτωσης στα πλαίσια κλινικών συνδρόμων ισχαιμίας. Παράλληλα, αναπτύχθηκαν και εφαρμόστηκαν καινοτόμες πειραματικές μεθοδολογίες για την πρόκληση, μορφολογική απεικόνιση και λειτουργική μελέτη της αγγειογένεσης και αρτηριογένεσης στα κάτω άκρα κονίκλων. Μελετήσαμε τις αρτηριογενετικές ιδιότητες της θρομβίνης σε ένα μοντέλο αμφοτερόπλευρης ισχαιμίας κάτω άκρων κονίκλου και δείξαμε για πρώτη φορά ότι η εξωγενής χορήγηση θρομβίνης ενισχύει το σχηματισμό νέων λειτουργικών και σταθερών παράπλευρων αγγειακών δικτύων και αποκαθιστά την αιματική άρδευση των ίσχαιμων κάτω άκρων. Η παρούσα μελέτη παρέχει τα πρώτα επιστημονικά δεδομένα για την αρτηριογενετική δράση της θρομβίνης, η οποία πιθανόν υπερέχει άλλων κλασσικών αγγειογενετικών παραγόντων (bFGF, VEGF). Επιπλέον εξετάσαμε διάφορα δοσολογικά σχήματα χορήγησης του μορίου της θρομβίνης και επιβεβαιώσαμε την αγγειογενετική της δράση τόσο σε ένα χειρουργικό, όσο και σε ένα ελάχιστα επεμβατικό ενδοαγγειακό μοντέλο αμφοτερόπλευρης ισχαιμίας των οπίσθιων άκρων του κονίκλου. Η επιτυχία της επαναιμάτωσης με θρομβίνη επαληθεύτηκε τόσο μορφολογικά με ψηφιακή αγγειογραφία όσο και λειτουργικά με σύγχρονα πρωτόκολλα υπολογιστικής τομογραφίας αιματώσεως. / Compared with angiogenesis, arteriogenesis is a distinct process based on the remodeling and maturation of pre-existing arterioles into large conductance arteries. Therapeutic angiogenesis has been proposed as a potential treatment for ischemic atherosclerotic diseases. Since a variety of angiogenic factors have been tested with inconsistent so far clinical results, the challenge remains in identifying the factor(s) that will stimulate functional neovascularization. Thrombin has been reported to play a pivotal role in the initiation of angiogenesis by regulating and organizing a network of angiogenic mediators. Also, it was recently demonstrated that thrombin is a potent anti-apoptotic factor for endothelial cells, providing evidence on a potential role of thrombin in vascular protection and maintenance of vessel integrity. Based on these observations, we hypothesized that thrombin may promote the development of mature functional blood vessels. We have shown that thrombin promoted the formation of large collateral vessels and improved the perfusion of distal ischemic tissue in a rabbit hindlimb ischemia model. These results provide new insights in understanding the involvement of thrombin in vascular formation and point to a novel role of thrombin in arteriogenesis. Interplay between angiogenic growth factors and vascular maturation mechanisms are essential for the cascade of reactions involved in arteriogenesis, i.e. development of large conductance collateral vessels that may adequately compensate for atherosclerotic arterial occlusions. Thrombin stimulated robust collateral networks in the ischemic limbs, which was associated with a significant recovery of ischemic tissue perfusion as assessed by in-vivo perfusion studies. This may provide the basis for applications of thrombin and its nonthrombogenic analogs in therapeutic angiogenesis.

No description available.