El remodelat vascular es un procés crític que present en el desenvolupament de patologies cardiovasculars com l’aterosclerosi i l’aneurisma d’aorta abdominal (AAA). La Lisil Oxidasa (LOX) i la Fibulina-5 (FBLN5) són dues proteïnes de matriu extracel•lular essencials en la formació i manteniment de les fibres elàstiques. Ambdues participen en processos fisiopatològics caracteritzats per l’alteració de l’equilibri síntesi/destrucció de la matriu extracel•lular i han estat involucrades en el remodelat vascular. Estudis en models experimentals han proporcionat indicis sobre la possible vinculació d’aquestes proteïnes en el desenvolupament d’aterosclerosi i d’AAA, si bé existeixen molts aspectes de la biologia d’aquestes proteïnes que haurien de ser analitzats en profunditat. Un aspecte crític en el remodelat vascular associat a la progressió de la lesió ateroscleròtica és l’aparició de regions d’hipòxia. L’estrès hipòxic és característic d’alguns processos fisiològics, però també de patologies com el càncer, els trastorns isquèmics, la inflamació crònica i l’aterosclerosi. Les cèl•lules endotelials són els sensors primaris d’aquest estrés hipòxic, i la seva adaptació a la hipòxia es produeix a través d’una complexa resposta finament regulada que afecta múltiples aspectes de la seva biologia, com la supervivència cel•lular, el control del to i la permeabilitat vascular, l’angiogènesi i el remodelat de la matriu extracel•lular. No obstant, no s’ha establert si la hipòxia pot regular la expressió de la LOX i la FBLN5 en cèl•lules endotelials ni la repercussió fisiològica que podria tenir. Les investigacions desenvolupades pel nostre grup indiquen que la LOX és un enzim clau en el manteniment de la integritat de l’endoteli i de la funció endotelial. La capacitat d’aquest enzim de controlar l’expressió gènica, en base a la seva activitat al nucli, així com l’activitat de factors de creixement fonamentals en l’homeòstasi vascular com el bFGF i el PDGF, suggereix que aquest enzim controla funcions cel•lulars que poden ser determinants en el desenvolupament de malalties cardiovasculars. En base a aquests antecedents, ens hem plantejat com a hipòtesis de treball que la LOX i la FBLN5 són proteïnes clau en l’homeòstasi vascular, que participen en la resposta adaptativa de les cèl•lules vasculars a la hipòxia, i que contribueixen al desenvolupament de patologies vasculars com l’aterosclerosi i l’aneurisma d’aorta abdominal. Els nostres resultats han demostrat que en artèries coronàries humanes, la LOX s’expressa principalment a l’endoteli i a l’adventícia, i que és la isoforma més expressada en cèl•lules vasculars, juntament amb la LOXL2. En cèl•lules endotelials, l’expressió de LOX i FBLN5 s’incrementa en resposta a hipòxia a través de mecanismes transcripcionals, si bé amb clares diferències entre ambdues proteïnes. Així, el factor HIF-1 juga un paper secundari en la regulació de la LOX per hipòxia en cèl•lules endotelials, una resposta en la que estan involucrades proteïnes de la família Smad i l’estrès oxidatiu. Per contra, HIF-1 és el principal responsable de la inducció de la FBLN5 en cèl•lules endotelials, en les quals hem demostrat que aquesta proteïna contribueix a la resposta adaptativa a l’estrès hipòxic. Finalment, hem observat que la sobre-expressió de LOX en cèl•lules endotelials mitjançant un sistema lentiviral indueix canvis en l’expressió gènica que afecten la senyalització i la comunicació cèl•lula-cèl•lula, d’entre els quals en destaca la inhibició de l’α2-macroglobulina. / Vascular remodeling is a critical process in the development of cardiovascular diseases such as atherosclerosis and abdominal aorta aneurysm (AAA). Lysyl oxidase (LOX) and Fibulin-5 (FBLN5) are two extracellular matrix proteins essential for the elastic fiber development and maintenance, and they both have an active role in physiological processes in which vascular remodeling is involved. By using different animal models, these proteins have been suggested to be involved with atherosclerosis and AAA. A critical aspect of the vascular remodeling in the atherosclerotic lesion is the apparition of hypoxic areas. Endothelial cells are primary sensors to the hypoxic stress, responding to it in a complex but subtly regulated way that affects multiple aspects of its biology. Nevertheless, it has not been established if the hypoxic stimulus can modulate LOX and FBLN5 expression, nor its possible physiological repercussions. Research made in our group shows the importance of LOX in the endothelial activity and vascular function. LOX can control the genetic expression as well as the activity of growth factors essential for the vascular homeostasis, suggesting that this enzyme may control cellular functions essential for the development of cardiovascular diseases. Based on these results, we have considered as working hypothesis that LOX and FBLN5 are key proteins in the vascular homeostasis, that they take part of the vascular response to hypoxia, and that they contribute to the development of vascular diseases like atherosclerosis and the AAA. Our results show that hypoxia transcriptionally induces LOX and FBLN5 in endothelial cells. While HIF-1 plays a secondary role in the regulation of LOX, being also involved in this modulation Smad proteins and oxidative stress, it is the main factor responsible for the FBLN5 induction. We also demonstrate that the modulation of FBLN5 in endothelial cells contributes to their adaptive response to hypoxia. Moreover, we have observed that overexpressing LOX in endothelial cells downregulates α2-macroglobulin expression.

The mechanical behavior of the arterial wall is determined by the composition and structure of its internal constituents as well as the applied traction-forces, such as pressure and axial stretch. The purpose of this work is to develop new theoretical frameworks and experimental methodologies to further the understanding of arterial mechanics and role of the various intrinsic and extrinsic mechanically motivating factors. Specifically, residual deformation, matrix organization, and perivascular support are investigated in the context of their effects on the overall and local mechanical behavior of the artery. We propose new kinematic frameworks to determine the displacement field due to residual deformations previously unknown, which include longitudinal and shearing residual deformations. This allows for improved predictions of the local, intramural stresses of the artery. We found distinct microstructural differences between the femoral and carotid arteries from non-human primates. These arteries are functionally and mechanically different, but are geometrically and compositionally similar, thereby suggesting differences in their microstructural alignments, particularly of their collagen fibers. Finally, we quantified the mechanical constraint of perivascular support on the coronary artery by mechanically testing the artery in-situ before and after surgical exposure.

The development of small diameter tissue engineered blood vessels (TEBVs) with low thrombogenicity, low immunogenicity, suitable mechanical properties, and a capacity to remodel to their environment could significantly advance the treatment of coronary and peripheral artery disease. Despite significant advances in the field of tissue engineering, autologous vessels are still primarily utilized as grafts during bypass surgeries. However, undamaged autologous tissue may not always be available due to disease or prior surgery. TEBVs lack long-term efficacy due to a variety of types of failures including aneurysmal dilations, thrombosis, and rupture; the mechanisms of these failures are not well understood. In vitro mechanical testing may help the understanding of these failure mechanisms. The typical mechanical tests lack standardized methodologies; thus, results vary widely. The overall goal of this study is to develop novel experimental and mathematical models to study the mechanical properties and failure mechanisms of TEBVs. Our results suggest that burst pressure tests, the current standard, are not sufficient to assess a TEBVs’ suitability as a coronary substitute; creep and/or cyclic loading tests are also required. Results from this model can help identify the most insightful experiments and quantities to be measured – ultimately reducing the overall number of experimental iterations. Improving the testing and characterization of TEBVs is critically important in decreasing the time necessary to validate the mechanical and functional responses of TEBVs over time, thus quickly moving TEBVs from the benchtop to the patient.

No description available.

This thesis explores two possible ways of detecting soft plaque present in the coronary arteries, using CTA imagery. The coronary arteries are vessels that supply oxidized blood to the cardiac muscle and are thus important for the proper functioning of heart. Cholesterol or reactive oxygen species from cigarette smoke and other toxins may get adhered to the walls of coronary arteries and trigger chronic inflammation that leads to formation of the soft plaque. When the soft plaque grows bigger in volume, it occludes the blood flow to the cardiac muscle and finally results in ischemic heart attack. Moreover, smaller plaque can easily rupture due to the blood flow in arteries and can result in complications such as stroke. Hence there is a need to detect the soft plaque using non-invasive or minimally invasive techniques. In CTA imagery, the cardiac muscle appears as a dark gray color, while the blood appears as dull white color and the the calcified plaque appears as bright white. The soft plaque has an intensity which falls between the intensity level of the blood and cardiac muscle, making it difficult to directly segment the soft plaque using standard segmentation methods. Soft plaque in its advanced stages forms a concavity in the blood lumen. A watershed based segmentation method was used to detect the presence of this concavity which in turn identifies the location of the soft plaque. For segmenting the soft plaque at its earlier stages, a novel segmentation technique was used. In this technique the surface is evolved based on a region-based energy calculated in the local neighborhood around each point on the evolving surface. This method seems to be superior to the watershed based segmentation method in detecting smaller plaque deposits.

Compliance mismatch is a significant hurdle to long-term patency in small-diameter arterial bypass grafts. Vascular tissue engineering has the potential to produce compliant, non-thrombogenic small-diameter grafts. However, current engineered grafts are relatively non-compliant, resulting in intimal hyperplasia and graft occlusion when subjected to arterial pressures. This research investigates the mechanical and biological properties of engineered constructs based on a biodegradable synthetic elastomer, poly(glycerol sebacate) (PGS). Several methods for fabricating porous PGS scaffolds in a tubular geometry were developed and compared. Adult baboon vascular cells were cultured in the scaffolds under various in vitro experimental conditions, including variations in initial cell seeding density, the type of scaffold used for culture, culture time, scaffold material, and hydrostatic pressure, and properties of the resultant constructs were compared. Scaffold fabrication using heat-shrinkable mandrels and glass tubes coated with hyaluronic acid significantly decreased tolerances of wall thickness and mechanical properties, improved handling, and decreased culture time required to reach luminal cellular confluence compared to scaffolds made with other fabrication techniques. Altering scaffold material from PGS to poly(lactide-co-glycolide) (PLGA), a benchmark biomaterial, did not affect scaffold yield, porosity, or luminal cellular confluence. Extracellular matrix (ECM) deposition increased with SMC-only culture time, and ECM deposition and remodeling during culture influenced construct compliance. Compared to PLGA scaffolds, PGS scaffolds promoted elastin crosslinking by SMCs and elastic tissue properties but attenuated collagen deposition. Hydrostatic pressure promoted ECM synthesis and deposition by SMCs and decreased construct compliance. Collagen and crosslinked elastin content in constructs correlated positively with construct burst pressure, and a negative correlation dependent on scaffold type was found between collagen content and construct compliance at low pressures. The systematic investigation of culture conditions in this research provides insights into the control of engineered blood vessel properties. The central hypothesis of this work, that grafts engineered from PGS scaffolds and adult vascular cells under biomimetic in vitro culture conditions can possess compliance comparable to autologous vessels, is true at pressures below 60 mmHg and demonstrates potential for PGS-based vascular tissue engineering. Overall, this work provides tools for engineering tubular soft tissues based on porous PGS scaffolds.

Vascular casts of three chondrichthian, one dipnoan, one chondrostean and 14 teleostean species were examined by light and scanning electron microscopy in order to give a qualitative and quantitative analysis of interarterial anastomoses (iaas) that indicate the presence (or absence) of a secondary vascular system (SVS). Anastomoses were found to originate from a variety of different primary blood vessels, many of which have not been previously identified as giving rise to secondary vessels. Segmental arteries derived from the dorsal aorta and supplying body musculature were major sites of origin of the SVS, although there was considerable variation in where, in the hierarchy of arterial branching, the anastomoses occurred. The degree of investment in a SVS was species specific, with more active species having a higher degree of secondary vascularisation. This difference was quantified using an absolute count of iaas between Anguilla reinhardtii and Trachinotus baillonii. A range of general features of the SVS is also described. No evidence of iaas was found on the coeliac, mesenteric or renal circulation in any species. Evidence of interarterial anastomoses were lacking in the dipnoan (Sarcopterygii) and chondrichthyan species examined, suggesting that a SVS is restricted to actinopterygian fishes. The presence and distribution of a secondary vascular system does not appear to be exclusively linked to phylogenetic position, but rather to the physiological adaptation of the species. Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel, Anguilla reinhardtii, were examined by light and transmission electron microscopy. Secondary vessels were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to re-anastomose with a secondary vessel running in parallel with the primary counterpart. Secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, surrounded by single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these were more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it was anticipated that flow through secondary vessels would to some extent be affected by an increase in primary vascular tone. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. Secondary vessels were followed to the surface of the animal through consecutive sections, where they eventually give rise to capillary beds overlying the scales. Secondary capillary beds were found to supply chloride cells in the skin, suggesting that this vascular system may be involved in cutaneous ionic exchange. Branchial vascular casts from two species of Tetraodontiformes showed that the vessels previously reported as nutrient vessels are with certainty part of the secondary vascular system. In the three-barred porcupine fish, Dicotylichthys punctulatus, interarterial anastomoses originated at high densities from efferent filamental and the efferent branchial arteries, from where they formed progressively larger secondary vessels. Small branches of this vascular system entered the filament body, where it gave rise to numerous side-vessels along the way. Large secondary vessels running in parallel with the efferent branchial arteries were found to constitute an additional arterio-arterial pathway, in that they exited the branchial basket in company with the afferent mandibular artery, the carotid artery and the efferent branchial arteries, from where they gave rise to vascular beds immediately after exit. The secondary vessels in this species were not found to supply the filament musculature; rather this vascular system was supplied by a single vessel derived from the efferent branchial artery, running in parallel with the afferent branchial artery. Secondary vessels were not found on any branchial component in the banded toadfish, Marylina pleurosticta, but in all other aspects the branchial vascular anatomy was similar to that of D. punctulatus. It is proposed that four independent vascular pathways may be present in the teleostean gill. The blood volume and flow rates of the primary (PVS) and secondary vascular system (SVS) were examined in the catadromous euryhaline teleost Lates calcarifer in order to determine whether any of these parameters were subject to change in individuals acclimated to seawater, compared to a group acclimated to freshwater. There was no significant difference in any measured parameter for the two groups. The volumes of the SVS were 0.67 „b 0.13 and 0.76 „b 0.13 mL 100g-1 body mass for FW and SW acclimated animals respectively. This constituted approximately one-third of the total blood volume in both groups. Turnover times for the SVS ranged from 21.0 to 25.2 minutes, demonstrating in accordance with previous publications, that this system is considerably more dynamic than previously assumed.

[Truncated abstract] Bone remodeling is an important process to maintain mechanical integrity. It is accomplished by two important steps, bone resorption followed by new bone formation. Osteoclasts and osteoblasts are the principal cells in bone resorption and bone formation, respectively. A multitude of local and systemic factors regulates this process by controlling the cellular activities in bone remodeling compartments (BRC). An imbalance of osteoblastic bone formation and osteoclastic bone destruction will result in the development of skeletal diseases. Recent studies suggested that angiogenesis is closely associated with bone remodeling. The vasculature in bone is important for skeletal development, growth and repair. During endochondral ossification, cartilage is invaded by blood vessels which bring in osteoblast and osteoclast precursor cells, nutrients, growth factors and differentiation factors. During fracture repair, it has been demonstrated that mature osteoclasts produce heparanase which can degrade heparin sulfate proteoglycans, a major component in extracellular matrix (ECM). The process leads to the release of heparin-binding growth factors including vascular endothelial growth factor (VEGF), a potent angiogenic factor which contributes largely to local angiogenesis. In recent studies, endothelial cells have been found to produce bone morphogenetic protein (BMP)-2 and BMP-4 when they are subjected to mechanical stimuli, or a hypoxia environment. Conversely, inhibition of angiogenesis has been shown to prevent fracture healing. In a distraction osteogenesis model, either inhibition of angiogenesis or disruption of the mechanical environment prevents normal osteogenesis and results in fibrous nonunion. .... A total of 42 mice from F1 and F2 generations were genotyped as transgene positive. Preliminary analysis using radiography did not reveal any difference between the gross structures of transgenic and wild type mice. Interestingly, the preliminary histology revealed a decrease in trabecular bone and an increase of lipid space in metaphysis of transgenic mice overexpressing EGFL6. However, further studies will need to be carried out to investigate the role of EGFL6 in angiogenesis and adipogenesis using a transgenic mice model. This will be a prime focus of future work. Collectively, the results presented in this thesis have identified EGFL6, a member of the EGF-like family, as a potential angiogenic factor which may play an important role in bone remodeling. EGFL6 has been found to be expressed highly in calvarial osteoblasts and upregulated during primary murine osteoblast differentiation. EGFL6 has been 8 characterized to be a secreted homomeric complex. More importantly, EGFL6 has been shown to induce angiogenic activity in endothelial cell migration, tube formation and in vivo chick embryo chorioallantoic membrane assay. Furthermore, conditioned medium containing the EGFL6 recombinant protein was shown to induce phosphorylation of ERK in endothelial cells. Inhibition of ERK impaired EGFL6-induced ERK activation and endothelial cell migration. Taken together these studies raise the possibility that EGFL6 has a potential role in angiogenesis, and mediates a paracrine mechanism of cross-talk between vascular endothelial cells and osteoblasts during osteogenesis. An understanding of this process offers the potential to facilitate the development of therapeutic treatments for bone disease.

Vascular casts of three chondrichthian, one dipnoan, one chondrostean and 14 teleostean species were examined by light and scanning electron microscopy in order to give a qualitative and quantitative analysis of interarterial anastomoses (iaas) that indicate the presence (or absence) of a secondary vascular system (SVS). Anastomoses were found to originate from a variety of different primary blood vessels, many of which have not been previously identified as giving rise to secondary vessels. Segmental arteries derived from the dorsal aorta and supplying body musculature were major sites of origin of the SVS, although there was considerable variation in where, in the hierarchy of arterial branching, the anastomoses occurred. The degree of investment in a SVS was species specific, with more active species having a higher degree of secondary vascularisation. This difference was quantified using an absolute count of iaas between Anguilla reinhardtii and Trachinotus baillonii. A range of general features of the SVS is also described. No evidence of iaas was found on the coeliac, mesenteric or renal circulation in any species. Evidence of interarterial anastomoses were lacking in the dipnoan (Sarcopterygii) and chondrichthyan species examined, suggesting that a SVS is restricted to actinopterygian fishes. The presence and distribution of a secondary vascular system does not appear to be exclusively linked to phylogenetic position, but rather to the physiological adaptation of the species. Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel, Anguilla reinhardtii, were examined by light and transmission electron microscopy. Secondary vessels were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to re-anastomose with a secondary vessel running in parallel with the primary counterpart. Secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, surrounded by single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these were more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it was anticipated that flow through secondary vessels would to some extent be affected by an increase in primary vascular tone. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. Secondary vessels were followed to the surface of the animal through consecutive sections, where they eventually give rise to capillary beds overlying the scales. Secondary capillary beds were found to supply chloride cells in the skin, suggesting that this vascular system may be involved in cutaneous ionic exchange. Branchial vascular casts from two species of Tetraodontiformes showed that the vessels previously reported as nutrient vessels are with certainty part of the secondary vascular system. In the three-barred porcupine fish, Dicotylichthys punctulatus, interarterial anastomoses originated at high densities from efferent filamental and the efferent branchial arteries, from where they formed progressively larger secondary vessels. Small branches of this vascular system entered the filament body, where it gave rise to numerous side-vessels along the way. Large secondary vessels running in parallel with the efferent branchial arteries were found to constitute an additional arterio-arterial pathway, in that they exited the branchial basket in company with the afferent mandibular artery, the carotid artery and the efferent branchial arteries, from where they gave rise to vascular beds immediately after exit. The secondary vessels in this species were not found to supply the filament musculature; rather this vascular system was supplied by a single vessel derived from the efferent branchial artery, running in parallel with the afferent branchial artery. Secondary vessels were not found on any branchial component in the banded toadfish, Marylina pleurosticta, but in all other aspects the branchial vascular anatomy was similar to that of D. punctulatus. It is proposed that four independent vascular pathways may be present in the teleostean gill. The blood volume and flow rates of the primary (PVS) and secondary vascular system (SVS) were examined in the catadromous euryhaline teleost Lates calcarifer in order to determine whether any of these parameters were subject to change in individuals acclimated to seawater, compared to a group acclimated to freshwater. There was no significant difference in any measured parameter for the two groups. The volumes of the SVS were 0.67 „b 0.13 and 0.76 „b 0.13 mL 100g-1 body mass for FW and SW acclimated animals respectively. This constituted approximately one-third of the total blood volume in both groups. Turnover times for the SVS ranged from 21.0 to 25.2 minutes, demonstrating in accordance with previous publications, that this system is considerably more dynamic than previously assumed.

Vascular casts of three chondrichthian, one dipnoan, one chondrostean and 14 teleostean species were examined by light and scanning electron microscopy in order to give a qualitative and quantitative analysis of interarterial anastomoses (iaas) that indicate the presence (or absence) of a secondary vascular system (SVS). Anastomoses were found to originate from a variety of different primary blood vessels, many of which have not been previously identified as giving rise to secondary vessels. Segmental arteries derived from the dorsal aorta and supplying body musculature were major sites of origin of the SVS, although there was considerable variation in where, in the hierarchy of arterial branching, the anastomoses occurred. The degree of investment in a SVS was species specific, with more active species having a higher degree of secondary vascularisation. This difference was quantified using an absolute count of iaas between Anguilla reinhardtii and Trachinotus baillonii. A range of general features of the SVS is also described. No evidence of iaas was found on the coeliac, mesenteric or renal circulation in any species. Evidence of interarterial anastomoses were lacking in the dipnoan (Sarcopterygii) and chondrichthyan species examined, suggesting that a SVS is restricted to actinopterygian fishes. The presence and distribution of a secondary vascular system does not appear to be exclusively linked to phylogenetic position, but rather to the physiological adaptation of the species. Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel, Anguilla reinhardtii, were examined by light and transmission electron microscopy. Secondary vessels were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to re-anastomose with a secondary vessel running in parallel with the primary counterpart. Secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, surrounded by single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these were more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it was anticipated that flow through secondary vessels would to some extent be affected by an increase in primary vascular tone. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. Secondary vessels were followed to the surface of the animal through consecutive sections, where they eventually give rise to capillary beds overlying the scales. Secondary capillary beds were found to supply chloride cells in the skin, suggesting that this vascular system may be involved in cutaneous ionic exchange. Branchial vascular casts from two species of Tetraodontiformes showed that the vessels previously reported as nutrient vessels are with certainty part of the secondary vascular system. In the three-barred porcupine fish, Dicotylichthys punctulatus, interarterial anastomoses originated at high densities from efferent filamental and the efferent branchial arteries, from where they formed progressively larger secondary vessels. Small branches of this vascular system entered the filament body, where it gave rise to numerous side-vessels along the way. Large secondary vessels running in parallel with the efferent branchial arteries were found to constitute an additional arterio-arterial pathway, in that they exited the branchial basket in company with the afferent mandibular artery, the carotid artery and the efferent branchial arteries, from where they gave rise to vascular beds immediately after exit. The secondary vessels in this species were not found to supply the filament musculature; rather this vascular system was supplied by a single vessel derived from the efferent branchial artery, running in parallel with the afferent branchial artery. Secondary vessels were not found on any branchial component in the banded toadfish, Marylina pleurosticta, but in all other aspects the branchial vascular anatomy was similar to that of D. punctulatus. It is proposed that four independent vascular pathways may be present in the teleostean gill. The blood volume and flow rates of the primary (PVS) and secondary vascular system (SVS) were examined in the catadromous euryhaline teleost Lates calcarifer in order to determine whether any of these parameters were subject to change in individuals acclimated to seawater, compared to a group acclimated to freshwater. There was no significant difference in any measured parameter for the two groups. The volumes of the SVS were 0.67 „b 0.13 and 0.76 „b 0.13 mL 100g-1 body mass for FW and SW acclimated animals respectively. This constituted approximately one-third of the total blood volume in both groups. Turnover times for the SVS ranged from 21.0 to 25.2 minutes, demonstrating in accordance with previous publications, that this system is considerably more dynamic than previously assumed.